Basic Research—Biology
The Effects of Antimicrobials on Endodontic Biofilm Bacteria
Luis E. Chávez de Paz, DDS, MS, PhD,* Gunnar Bergenholtz, DDS, PhD,†
and Gunnel Svensäter, DDS, PhD*
Abstract
Introduction: In the present study, confocal microscopy,
a miniflow cell system, and image analysis were combined
to test in situ the effect of antimicrobials and alkali on bi-
ofilms of Enterococcus faecalis, Lactobacillus
paracasei, Streptococcus anginosus, and Strepto-
coccus gordonii isolated from root canals with persis-
tent infections. Methods: Biofilms formed for 24 hours
were exposed for 5 minutes to alkali (pH = 12), chlorhex-
idine digluconate (2.5%), EDTA (50 mmol/L), and sodium
hypochlorite (1%). The biofilms were then characterized
by using fluorescent markers targeting cell membrane
integrity (LIVE/DEAD) and metabolic activity (5-cyano-2,3-
ditolyl tetrazolium chloride and fluorescein diacetate). In
addition, the biofilm architecture and the extent to which
coating of the substrate surface with collagen influenced
the resistance pattern to the chemicals were also analyzed.
Results: NaOCl (1%) affected the membrane integrity of
all organisms and removed most biofilm cells. Exposure
to EDTA (50 mmol/L) affected the membrane integrity in
all organisms but failed to remove more than a few cells
in biofilms of E. faecalis, L. paracasei, and S. angino-
sus. Chlorhexidine (2.5%) had a mild effect on the
membrane integrity of E. faecalis and removed only
50% of its biofilm cells The effects were substratum-
dependent, and most organisms displayed increased resis-
tance to the antimicrobials on collagen-coated surfaces.
Conclusions: The biofilm system developed here was
sensitive and differences in cell membrane integrity and
removal of biofilm cells after exposure to antimicrobials
commonly used in endodontics was discernible. (J Endod
2010;36:70–77)
Key Words
Alkaline resistance, antimicrobial resistance, biofilm
model, biofilm system, biofilms, environmental changes,
image analysis, microbial adaptation, persistent infec-
tions, substrate conditioning
From the *Department of Oral Biology, Faculty of Odontol-
ogy, Malmö University, Malmö, Sweden; and †Department of
Endodontology, The Sahlgrenska Academy at The University
of Gothenburg, Gothenburg, Sweden.
Address requests for reprints to Dr Luis E. Chávez de Paz,
Department of Oral Biology, Faculty of Odontology, Malmö
University, Malmö SE-20506, Sweden. E-mail address: luis.
chavez.de.paz@od.mah.se.
0099-2399/$0 - see front matter
Copyright ª 2010 by the American Association of
Endodontists. All rights reserved.
doi:10.1016/j.joen.2009.09.017
70 Chávez de Paz et al.
T he proposed efficacy of chemicals with antimicrobial properties for disinfection in
endodontics has often been based on their activity against microorganisms grown in
liquid cultures in vitro. Test systems using planktonic cells do not mirror the conditions
in vivo because the microorganisms to be targeted rather than being dispersed are
likely to be organized in structures attached to each other and/or the root canal walls
often involving a multitude of species known as microbial biofilms. It is now well recog-
nized that such microbial communities are noticeably resistant to and difficult to erad-
icate with antimicrobials (1, 2). Deep-seated microorganisms, in particular, may
escape killing and remain viable for remultiplication when conditions are favorable
and, thus, cause an endodontic treatment failure. A recent in vitro study (3) showed
that wild-strain bacteria of endodontic origin grown over 8 days on dentin slices of
extracted teeth to generate biofilms were not possible to eradicate using ampicillin,
doxycycline, clindamycin, azithromycin, or metronidazole.
Various techniques for the effective control of endodontic biofilm infections are
being explored. Promising in vitro data have been reported for photodynamic therapy
(4–7) as well as ultrasonic irrigation performed with small files or wires that are acti-
vated to oscillate freely in the root canal space and mechanically dislodge experimental
biofilms (8, 9). Also, the effects of endodontic irrigants have been tested on root canal
bacteria in vitro by growing biofilms on extracted teeth (10), a cellulose nitrate
membrane placed on agar medium (11), and membrane filter disks (12).
Studying endodontic microorganisms when adhered to surfaces and how they may
respond to antimicrobials calls for relevant in vitro models. Thus far, the testing of anti-
microbial agents against bacteria in biofilms has not been standardized. Some studies
have used models that include the mechanical removal of biofilm cells followed by tradi-
tional cell cultures for enumeration of survivors (13, 14). Alternative approaches
include the use of confocal microscopy combined with fluorescent viability staining
for in situ investigation of antimicrobial effects on biofilms (15). In the present study,
we use confocal microscopy in combination with a miniflow cell system and image anal-
ysis to examine biofilm responses in situ to alkaline pH and some common disinfectants
used in endodontics including chlorhexidine, EDTA, and sodium hypochlorite. In addi-
tion, we also analyze the extent to which collagen coating of the substrate surfaces might
influence the biofilm resistance to these disinfectants. A structural and metabolic char-
acterization of the biofilms formed by four individual root canal isolates is presented.
Material and Methods
Bacterial Strains and Culture Conditions
The bacterial strains used in the present study, Enterococcus faecalis, Lactoba-
cillus paracasei, Streptococcus anginosus, and Streptococcus gordonii, were iso-
lated from root canals undergoing endodontic treatment in patients who presented
with persistent infections (16). Strains were stored at 70 C in skim milk powder
(Oxoid, Hampshire, UK) diluted in double distilled water. Strains were recovered on
blood agar in an atmosphere of 5% CO2 in hydrogen at 37 C for 24 hours.
To prepare the inoculum, colonies from blood agar were transferred into Todd-He-
witt (TH) liquid growth medium and incubated in an atmosphere of 5% CO2 in hydrogen at
37 C overnight. Aliquots (500 mL) were transferred to 10 mL of fresh TH and incubated at
37 C; the optical density at 600 nm wavelength (OD600) was monitored until the exponen-
tial growth phase was reached (ie, OD600 values of 0.6  0.1). Cells were harvested by
centrifugation (3,000g, 5 minutes at 4 C), washed in 10 mmol/L phosphate-buffered
saline (PBS), and resuspended in TH to an approximate cell concentration of 1  108/mL.
JOE — Volume 36, Number 1, January 2010
 Basic Research—Biology
100 100
2.5% CLX Alkali (pH12)

75 75
% survivors

% survivors
50 50

25 25

0 0
Ef Lp Sa Sg Ef Lp Sa Sg

100 100
50mM EDTA 1% NaOCl

80
75
% survivors
% survivors

60

50

40

25
20

0 0
Ef Lp Sa Sg Ef Lp Sa Sg

Figure 1. The survival of biofilm cells after 5 minutes of exposure to chlorhexidine digluconate (2.5%), alkali (pH 12), EDTA (50 mmol/L), and NaOCl (1%).
E. faecalis (Ef), L. paracasei (Lp), S. anginosus (Sa), and S. gordonii (Sg).

Biofilm Formation
Biofilms were created in miniflow chamber systems m-Slide VI for
Live Cell Analysis (Integrated BioDiagnostics, Munich, Germany) with
hydrophilic uncoated surfaces and with surfaces conditioned with
collagen type IV. Biofilms were created as described in a previous study
(17). Briefly, each flow chamber was inoculated with 30 mL of a washed
suspension of cells grown to the midexponential phase in TH, followed
by the addition of 100 mL of fresh TH to give a final volume of 130 mL per
flow chamber. Flow chambers were incubated in an atmosphere of 5%
CO2 in hydrogen at 37 C. After 24 hours of biofilm growth, chambers
were rinsed with phosphate-buffered saline (PBS) (pH = 7.4) to
remove nonadherent cells.
sessed. The universal protocol was to aspirate the TH medium and to add
40 mL of the antimicrobial test solution. The samples were left at room
temperature for 5 minutes, and cell membrane integrity was assessed by
using the BacLight Bacterial Viability kit for microscopy (LIVE/DEAD)
(Invitrogen Ltd, Paisley, UK) and image analysis as described later.
Fluorescent Staining for Cell Membrane Integrity
Cell samples were stained separately for dehydrogenase activity,
esterase activity, and membrane integrity with fluorescent dyes. Three
triplicate sets of biofilms formed in noncoated surfaces and on surfaces
coated with collagen type IV were cultured under the same conditions
with each set having its own unique total cell count.

Resistance to Alkali and Antimicrobials Membrane integrity

In order to test the dependence of substratum and whether resis-
tance changed with respect to antimicrobials, biofilms established for 24
hours were exposed for 5 minutes to PBS adjusted to pH = 12 with
NaOH, NaOCl (1%), chlorhexidine digluconate (2.5%), or EDTA
(50 mmol/L), respectively. Thereafter, cell membrane integrity was as-
LIVE/DEAD stain was used to assess membrane integrity of biofilm
cells. The LIVE/DEAD mixture was prepared by mixing components A +
B in the kit, consisting of SYTO9 and propidium iodide, at a ratio of
1:100 with the inoculum medium (TH). After removing the culture fluid
from the flow chamber and washing with PBS, 30 mL of the LIVE/DEAD

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Basic Research—Biology

Figure 2. Three-dimensional reconstructions from confocal micrographs showing the removal of biofilm cells by antimicrobials using LIVE/DEAD staining. The
green cells represent cells with intact membranes, whereas the red cells are damaged or dead. The left-hand column shows the controls with their corresponding
biovolumes (BV). The percentage of cells removed in relation to the controls is presented below each image. Images were reconstructed using the software
bioImage_L (18).

mixture was added. Chambers were incubated at room temperature for
10 minutes. The fluorescence from stained cells was viewed by using
confocal scanning laser microscopy (CSLM) (described later).
Dehydrogenase activity
The redox fluorescence dye, 5-cyano-2,3-ditolyl tetrazolium chlo-
ride (CTC) (Invitrogen Ltd, Paisley, UK), which targets dehydrogenase
activity, was used to assess the physiologic status of the test bacteria.
After removing the culture fluid and washing with PBS, 30 mL CTC
(5 mmol/L) was added to the chamber. To visualize nonactive cells, bi-
ofilm samples were counterstained with 1 mL of 1 mmol/L SYTO 24
(green fluorescence) (Invitrogen Ltd). Fluorescence of the stained cells
was examined by CSLM.
Esterase activity
Fluorescin diacetate (FDA) (Invitrogen Ltd), which reacts with
nonspecific esterases in metabolically active microorganisms, was
also used as a metabolic marker. A stock solution of FDA was prepared
by mixing 5 mg FDA/mL in pure acetone. The working solution was
prepared by mixing 5 mL FDA/acetone/mL in TH medium. After
removing the culture fluid from the biofilm chamber and washing
with PBS, 30 mL FDA working solution was added. Ethidium bromide

72 Chávez de Paz et al. JOE — Volume 36, Number 1, January 2010

 Basic Research—Biology
TABLE 1. Survival of Biofilm Cells on Collagen-coated Surfaces after 5 Min Exposure to Alkali and Antimicrobials. Values are Mean % of Survivor Cells  SE
of Triplicate Experiments
Control CLX (2.5%) pH 12 EDTA (50 mM) NaOCl (1%)
E. faecalis 98  1 22  4 97  1 11  2 26  8
L. paracasei 77  4 83 77  2 0 30  4
S. anginosus 84  3 44  3 64  5 51  1 2  0.5
S. gordonii 78  4 58  4 52  4 42  7 53  5

(EB) was used as a counterstain to visualize nonactive cells. The EB
stock solution was prepared by mixing 0.5 mg EB (Carl Roth GmbH,
Karlsruhe, Germany) per milliliter in NaCl. The working solution was
a 1:200 dilution. EB was added in 20-mL aliquots to the biofilm cham-
bers already containing FDA. The biofilm chambers were incubated in
the conditions described previously. Fluorescence of the stained cells
was examined by CSLM.
Confocal Scanning Laser Microscopy
All microscopy work was performed by using an Eclipse TE2000
inverted confocal scanning laser microscope (Nikon, Tokyo, Japan). By
means of a motorized stage, 10 randomly selected image stack sections
were imaged in each biofilm sample. Image stack sections were
composed of 10 images, each taken with a variation of 2 mm along
the z-position. Images were obtained by using a 60 magnification
oil immersion objective with a numeric aperture of 1.4 and the confocal
pinhole set to a diameter of 30 mm. In all cases, CSLM images were
acquired by the software EZ-C1 v.3.40 build 691 (Nikon) at a resolution
of 512 pixels2 and with a zoom factor of 1.0, giving a final pixel reso-
lution of 0.41 mm/pixel. Individual biofilm images covered an area of
0.05 mm2 per field of view.
For the LIVE/DEAD and FDA/EB techniques, confocal illumination
was provided by an Ar laser (488-nm laser excitation) fitted with a long-
pass 515/30 filter for the green fluorescence signal (emitted by SYTO9
and FDA) and a long-pass 605/75 filter for the red fluorescence signal
(propidium iodide and EB). Simultaneous dual-channel imaging was
used to display green and red fluorescence. For detection of the red
fluorescence by CTC staining, a G-HeNe laser was used (543-nm laser
excitation), together with a long-pass 605/75 emission filter. SYTO 24
was detected by the same laser with an emission filter of 515/30 in order
to determine the numbers of cells not reacting with the CTC stain.
Image Analysis of Microbial Biofilms
CSLM images were analyzed in two dimensions and three dimen-
sions by using the software bioImage_L (18). The bioImage_L software
is based on color segmentation algorithms written in MATLAB (The
MathWorks, Natick, MA), which produce information of the total bio-
film population as well as the independent subpopulations represented
by red and green fluorescent colors. Structure and distribution of bio-
films are characterized by the following variables: biovolume (mm3),
TABLE 2. Removal of Biofilm Cells on Collagen-coated Surfaces after 5 Min
Exposure to Alkali and Antimicrobials. Values Are Mean % of Removed Cells 
SE of Triplicate Experiments
EDTA NaOCl
CLX (2.5%) pH 12 (50 mM) (1%)
E. faecalis 53  1 15  1 57  2 83  1
L. paracasei 28  3 51 20  2 78  1
S. anginosus 41  2 69  2 11  1 89  3
S. gordonii 28  3 50  4 57  1 88  2
substratum coverage (%), and mean biofilm height (mm). Biovolume
represents the overall volume of the biofilm including an estimate of
the biomass in the biofilm, substratum coverage reflects how efficiently
the organism colonizes the substratum, and mean height indicates the
spatial thickness of the biofilm. To visualize structure and spatial differ-
ences, three-dimensional reconstructions were produced by the func-
tion ‘‘structure and distribution’’ in bioImage_L.
The percentage (%) survival of biofilm cells determined by the
LIVE/DEAD technique was obtained by calculating the percentage of
the biovolume of the green subpopulation from the total biovolume
of the biofilm. Accordingly, the percentage of metabolic active cells in
biofilms after staining with CTC was obtained by calculating the propor-
tion of red cells (metabolic active) in relation to the total biovolume.
The removal of biofilm cells was calculated by comparing the biovolume
of biofilms after the antimicrobial exposure with the biovolumes of
biofilms before treatment.
Statistical Analysis
To confirm that the patterns observed were reproducible, each
experiment was repeated three times. Hence, results are presented as
mean calculations from a total of 30 biofilm sections (ie, 10
sections/flow-chamber channels in three independent experiments).
The software bioImage_L also included a function that analyzed the
three independent biofilms by two-way analysis of variance. This statis-
tical analysis detected any significant variation in terms of time, varia-
tions among the color-base–identified subpopulations, interaction
between surface coating, and random variation (error).
Results
Biofilm Survival to Alkali and Antimicrobials
E. faecalis, L. paracasei, S. anginosus, and S. gordonii biofilms
grown over 24 hours on polystyrene flow-cell chambers were used for
testing the effect of alkali (pH = 12), chlorhexidine diclugonate (2.5%),
EDTA (50 mmol/L), and sodium hypochlorite (1%). The cell
membrane integrity of biofilm cells after 5 minutes of exposure to all
four compounds was determined by LIVE/DEAD staining and image
analysis. As shown in Figure 1, exposure to 2.5% chlorhexidine diclug-
onate had a large effect on streptococcal biofilms, with only 20%  3%
and 21%  2%, respectively, of the S. anginosus and S. gordonii cells
surviving. The lowest effect of chlorhexidine was observed for E. faeca-
lis and L. paracasei with 60%  2% and 30%  1%, respectively, of the
cells surviving.
After exposure to alkali (pH = 12), biofilms of S. anginosus and
S. gordonii showed 50%  2% and 51%  4%, respectively, of the cells
surviving. The same stress condition failed to damage more than 10% of
the E. faecalis and L. paracasei biofilm cells. EDTA (50 mmol/L) and
sodium hypochlorite (1%) had the greatest effect on cell membrane
integrity, producing up to 100% damaged cells in the biofilm popula-
tions of the four species tested.

JOE — Volume 36, Number 1, January 2010 Effects of Antimicrobials on Endodontic Biofilm Bacteria 73
Basic Research—Biology

Figure 3. The effect of surface coating with collagen on biofilm structure. Images are three-dimensional reconstructions from confocal micrographs. Biofilm
sections were artificially colored blue for better visualization of the architecture and reconstructed by using the software bioImage_L (18). Scale units, mm.

74 Chávez de Paz et al. JOE — Volume 36, Number 1, January 2010

 Basic Research—Biology
Removal of Biofilm Cells by Means of Alkali and A 100
Antimicrobials
The effect of chlorhexidine, pH12, EDTA, and NaOCl was tested on

% CTC active biofilm cells

the removal of biofilm cells in the aforementioned organisms (Fig. 2).
75
The most efficient removal action was achieved by sodium hypochlorite
that removed 91%  1% of E. faecalis, 84%  2% of L. paracasei,
76%  3% of S. anginosus, and 71%  1% of S. gordonii biofilms.
Chlorhexidine removed a large number of cells in biofilms of S. angi- 50
nosus (79%  3%) and E. faecalis (49%  4%), whereas pH = 12
showed a significant removal effect only in S. anginosus biofilms
(63%  6%). Similarly, EDTA was effective in removing S. gordonii 25
(83%  3%) and S. anginosus (33%  1%) biofilm cells.

Influence of Surface Coating with Collagen on Biofilm

Resistance 0
Ef Lp Sa Sg
In order to assess whether the observed effects of the disinfectants
would be influenced by surface conditioning, the four root canal
bacteria were allowed to form biofilms on collagen-coated flow cells B 100
for 24 hours and were exposed to the disinfectants. The results on
bacterial survival and removal of biofilm cells are shown in

% Esterase active biofilm cells

Table 1and 2, respectively.
In contrast to the results observed on noncoated surfaces, biofilms
of S. gordonii and S. anginosus showed the highest resistance to
chlorhexidine with 58%  4% and 44%  3% of the cells surviving,
respectively. E. faecalis and L. paracasei biofilms had the lowest resis-
tance to this antimicrobial. The removal effect on S. anginosus biofilms
was reduced to 41%  2%.
Exposure to pH = 12 buffer had no significant effect on biofilms of
E. faecalis, L. paracasei, and S. gordonii as compared with noncoated
surfaces (p < 0.001). However, S. anginosus showed a significant
increase in surviving cells with 64%  5%. Although exposure to
EDTA had a strong effect on membrane integrity for all four organisms
on noncoated surfaces, it was less effective on streptococcal biofilms on
collagen-coated surfaces. Streptococcal biofilms were also not as effec-
tively removed as their non-coated counterparts. In a similar fashion, in
biofilms of E. faecalis, L. paracasei, and S. gordonii, the number of
survivors after the exposure to NaOCl was significantly increased.
Influence of Surface Coating with Collagen on Biofilm
Structure
The observed substratum-dependent differences in biofilm resis-
tance to antimicrobials can be explained by differences in biofilm
structure and distribution. Preconditioning the flow-cell surfaces
with collagen noticeably modified the biofilm mass volume (biovo-
lume, mm3) and percentage substratum coverage by the four microor-
ganisms compared with those formed on uncoated surfaces (Fig. 3).
All four organisms formed unevenly distributed biofilms on the
substrate with reduced substratum coverage and biovolumes on
collagen-coated surfaces. This trend was clearly seen in S. anginosus
and S. gordonii, which presented two-fold lower biovolumes and less
substratum coverage as compared with biofilms formed on uncoated
surfaces.
Effect of Surface Conditioning on Metabolic Activity
of Biofilms
To further characterize the differences between surfaces, meta-
bolic activity was tested by means of the redox fluorescence dyes CTC
and FDA, which target dehydrogenase active cells and cells producing
esterases, respectively. Figure 4A shows mean values of metabolic active
biomass (%) of biofilm cells reducing CTC, and Figure 4B shows the
75
50
25
0
Ef Lp Sa Sg
Figure 4. The effect of surface coating with collagen on cell metabolic activity.
(A) The effect on dehydrogenase activity and (B) the effect on esterase activity.
White bars: noncoated surfaces and gray bars: surfaces coated with collagen.
proportion of FDA active cells. Significant reductions in dehydrogenase
activity and esterase activity were evident for all species in biofilms on
collagen-conditioned surfaces. For E. faecalis and L. paracasei, the
reduction in CTC active cells was six-fold and four-fold, respectively.
For S. anginosus and S. gordonii, these levels were 12- and 13-fold,
respectively. Esterase activity was reduced in all organisms.
Discussion
In this study, we introduced a system for analyzing the effect of anti-
microbials in biofilms, which is based on in situ examination of bacte-
rial cell membrane integrity and biofilm structure and metabolic activity
using fluorescent staining and image analysis. By means of this system,
we were able to observe differences in membrane integrity, metabolic
activity, and removal of biofilm cells after treatment with antimicrobials
commonly used in endodontics. Although it is realistic to acknowledge
that no in vitro method will accurately reflect the conditions under
which microorganisms grow in vivo, the implementation of models
that take into consideration that endodontic organisms are likely to
prevail in communities attached to the root canal walls should better
our understanding of their persistence and how they may be combated
clinically.
Others have assessed cell membrane integrity by mechanical
removal of cells and enumeration through traditional culture (7, 13,

JOE — Volume 36, Number 1, January 2010 Effects of Antimicrobials on Endodontic Biofilm Bacteria 75
Basic Research—Biology
19). However, such methods have inherent errors that are connected
with incomplete cell removal, resulting in poor homogeneity of the
culture suspension. Furthermore, membrane integrity assessment of
removed biofilm cells might be hampered by the presence of cells in
metabolically inactive states, which are incapable of cellular division
preventing them from forming colonies on a plate (17, 20). This may
lead to overestimation of the efficacy of the tested antimicrobials.
In the present study, NaOCl (1%) and EDTA (50 mmol/L) seemed
to be the most effective of the agents tested by affecting the cell
membrane integrity of all organisms in biofilms on uncoated surfaces.
Although EDTA is typically considered as a ‘‘nonantibiotic’’ agent and
used in endodontics to remove the smear layer for improved sealing
during root filling (21), recent studies have shown antimicrobial effects
of EDTA against microorganisms in biofilms (22, 23). Although the
mode of action of EDTA in this respect is still not clarified, the chelating
effect to calcium and iron may affect important metabolic pathways in
the bacterial cell (24).
In the present study, the highest antimicrobial effects of chlo-
rhexidine were observed in streptococcal biofilms. This finding is in
accordance with a recent study in which high antimicrobial effects
of chlorhexidine were shown in 3-week-old biofilms formed from
subgingival plaque and grown on hydroxyapatite surfaces (25).
However, it is interesting to note that the effect of chlorhexidine
was not as high in the overall biovolume of biofilms by L. paracasei
and E. faecalis. In these organisms, we observed that cells in the
upper layers of the biofilms were more affected by chlorhexidine
than those in deeper layers (data not shown). Such limited penetra-
tion of chlorhexidine has also been reported to occur in dental plaque
biofilms in which chlorhexidine showed the highest antimicrobial
effect in the outermost layers of dental plaque but failed to kill cells
deeper in the biofilm (15).
Alkali showed the least antimicrobial effect on biofilms of root canal
bacteria. As indicated in previous reports, tolerance to alkali, and
possibly other agents, is likely to be connected to the expression of
phenotypes resistant to these agents within the biofilm communities (26).
The removal of biofilm cells was effectively achieved by NaOCl in all
organisms tested. Although EDTA and alkali removed biofilm cells effec-
tively in S. anginosus biofilms, chlorhexidine removed most cells in
S. gordonii biofilms. EDTA and alkali may have similar actions to break
up the polymeric matrix structure of biofilms in which they may sequester
divalent ions essential for cell coadherence. On the other hand, chlorhex-
idine targets negatively charged molecules, interferes with enzyme inhi-
bition, and precipitates proteins and nucleic acids (27). Although the
cell-removing selectiveness of EDTA, alkali, and chlorhexidine remains
unclear, it is likely that the diffusion of these agents is time dependent,
and 5 minutes may not be long enough to penetrate biofilms of organisms
that may have formed matrix with different densities and components.
Our findings show that biofilm structure and susceptibility to anti-
microbials is affected by a number of factors such as the surrounding
nutrient environment and the substratum. The substratum will not
only influence the initial adhesion of colonizing cells, but it will also
influence the production of signaling molecules that control cell phys-
iology and virulence. In the present study, biofilms formed on surfaces
preconditioned with collagen showed a more patchy structure than
those formed on clean polystyrene surfaces (Fig. 3). Such a varying
distribution may be explained by a selection of cells that adhere exclu-
sively on the weakly hydrophobic tracks created by surface oxidation on
the collagen-substratum interface (28). Apparently, such phenomena
occurring at the collagen-substratum interface level may influence the
stress response in biofilm bacteria when exposed to antimicrobials.
For instance, S. gordonii, E. faecalis, and L. paracasei showed
a much higher number of viable cells after exposure to NaOCl, although
76 Chávez de Paz et al.
the proportion of removed cells was still high. The mechanisms behind
these changes are not fully understood, and, therefore, we undertook an
in situ screening of metabolic activity. The levels of dehydrogenase and
esterase activities of biofilm cells on collagen-coated surfaces were
much lower than on noncoated surfaces. This metabolic down-
regulation may give some indications as to how the surface condition
may influence bacterial physiology.
The biofilm analysis method presented in this report, including the
assessment of both cell membrane integrity and biofilm structure,
provides a broad picture of the in situ effectiveness of antimicrobials
against bacteria in biofilms. After controlling variables such as substratum
conditioning, the presented system can be used to monitor not only vari-
ations of structure in biofilms, such as biovolume and area coverage, but
also the distribution of undamaged cells with active metabolism in three
dimensions. This biofilm model seems useful for assessment of the
efficacy of a variety of agents used for root canal disinfection.
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