Basic ResearchBiology

The Resistance of Collagen-associated, Planktonic Cells of

Enterococcus faecalis to Calcium Hydroxide
Guven Kayaoglu, DDS, PhD,* Hulya Erten, DDS, PhD,* Emre Bodrumlu, DDS, PhD, and
Dag rstavik, DDS, PhD,
Abstract
This study examined whether collagen association by
an endodontic isolate of Enterococcus faecalis conferred resistance to the bacterium against calcium hydroxide. E. faecalis A197A was grown at 46C until
early stationary phase. Standardized bacterial suspensions were pretreated for 1 hour either with acidsoluble collagen or acidified phosphate-buffered saline
(ac-PBS) and cultured to determine the baseline viable
bacterial numbers. The bacterial suspensions were challenged with calcium hydroxide solution. Samples were
removed at 6, 12, and 24 hours and cultured on tryptone soy agar plates. An adherence assay was performed to confirm that the collagen in the pretreatment
medium was bound by the bacteria. Significantly more
bacteria were cultivated at 12 hours in the collagenpretreated group than the ac-PBSpretreated group
(p 0.01). No bacteria could be cultivated at 24 hours
in either group. Collagen association by E. faecalis
A197A was found to increase the tolerance of the
bacterium to calcium hydroxide. (J Endod 2009;35:
46 49)

Key Words
Adhesin, adhesion, binding, disinfection, susceptibility

From the *Department of Endodontics and Conservative

Treatment, Faculty of Dentistry, Gazi University, Ankara, Turkey; Department of Operative Dentistry and Endodontics,
Ondokuz Mayis University, Samsun, Turkey; and Institute for
Clinical Dentistry, University of Oslo, Oslo, Norway.
Supported by The Research Council of Norway and NIOM
Scandinavian Institute of Dental Materials.
This study was part of the doctorate thesis by Guven
Kayaoglu: Investigation of infection and survival mechanisms
held by Enterococcus faecalis (strain A197A): with respect to
endodontic disease, Gazi University, Ankara, 2007.
Address requests for reprints to Dr Guven Kayaoglu, Department of Endodontics and Conservative Treatment, Gazi
University, 82.sokak, 06510, Emek, Ankara, Turkey. E-mail
address: guvenk@gazi.edu.tr.
0099-2399/$0 - see front matter
Copyright 2008 American Association of Endodontists.
doi:10.1016/j.joen.2008.09.014

46

Kayaoglu et al.

nterococcus faecalis is a gram-positive, facultative anaerobic bacterium that can

cause serious clinical diseases including endocarditis, bacteremia and urinary tract
infections and is among the major nosocomial pathogens (1). E. faecalis is also one of
the most prevalent species found in persistent root canal infections refractory to endodontic treatment (2 4).
Adhesion to and colonization of the host by microorganisms are the first steps in
the establishment of most infectious diseases, after which pathological changes occur.
In endodontic infection, dentinal tubule colonization by E. faecalis has been shown to
occur through adhesion of the bacterium to collagen, the main organic component of
the dentine (5). E. faecalis possesses a collagen-binding proteinaceous adhesin named
Ace, which is produced by the bacterium at stressful growth conditions (eg, during
growth at 46C) or during human infections (6, 7). Ace is an important factor in the
adhesion of E. faecalis to dentine (8).
In the planktonic state, E. faecalis can be eliminated rapidly using disinfectants (9,
10). However, when dentine is included in the experimental design, it can hardly be
eliminated (11, 12). This can be attributed partly to interactions between dentine and
the disinfectant because dentine has a buffering effect on disinfectants (12). The interaction between dentine and E. faecalis may also render the bacterium resistant. E.
faecalis adhering to dentinal surfaces is more resistant against calcium hydroxide
compared with its free-floating counterparts (13). We have previously found that collagen-associated E. faecalis may be more resistant against chlorhexidine and iodine
potassium iodide but more susceptible to calcium hydroxide compared with bacteria
not exposed to collagen (14). Because increased susceptibility to calcium hydroxide
was an unexpected finding, the latter study addressed the need for further studies on the
effect of collagen association by E. faecalis to its resistance against calcium hydroxide.
Previous studies have shown that an endodontic isolate of E. faecalis (strain A197A)
possesses the ace gene and adheres to collagen at increased numbers after growth at
46C, an established stressful growth condition (15, 16). This study sought to assess
whether association with collagen conferred resistance on E. faecalis A197A against
calcium hydroxide.

Materials and Methods

Bacterial Strain and Culture Conditions
The bacterial strain used in this experiment was E. faecalis A197A, a clinical
isolate from a persistent endodontic infection (17). Tryptone soy broth (TSB; Oxoid Ltd,
Basingstoke, England) was used as the growth medium. Glycerol stock of the bacteria
was prepared by growing cells overnight in TSB at 37C. The cells were harvested by
centrifugation (10,000g, 10 minutes, 4C) and resuspended in fresh TSB containing
40% (v/v) glycerol as a cryoprotectant. This stock suspension was stored at 20C
until required.
A 2% inoculum from the glycerol stock was grown in fresh TSB at 46C to the early
stationary phase. The cells were centrifuged 3 times (10,000g, 10 minutes, 4C),
washed with phosphate-buffered saline (PBS, pH 7.4), and finally resuspended in PBS.
The bacterial density was standardized using a spectrophotometer (Multiskan EX; Labsystems, Helsinki, Finland) to OD540 1.0, which normally corresponds to 1 2
109 bacteria per mL. Pilot experiments had shown that viable bacterial numbers for
46C- and 37C-grown bacteria were similar.

JOE Volume 35, Number 1, January 2009

Downloaded from ClinicalKey.com at N K P Salve Institute Of Medical Sciences and Research Centre July 19, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.

Basic ResearchBiology

Figure 1. The standard curve constructed by intersecting the known amount of

bacteria and OD readings at 570 nm.

Pretreatment Conditions
One thousand fifty-microliter aliquots of the bacterial suspensions
were added to plastic test tubes. In group 1, 1,050 L of acid-soluble
collagen type I solution in PBS (100 g/mL) (Sigma Chemical Co, St
Louis, MO) was added. The pH of the collagen solution at this concentration was 4.25, as measured using a digital pH meter. The resultant
concentration of collagen in the mixture with bacteria was 50 g/mL.
The pH of the mixture was 4.91. In group 2, 1,050 L of acidified PBS
(ac-PBS) was added, the pH of which had been adjusted to 4.25 by
addition of acetic acid. There were four parallel tubes in each group.
The mixtures were then incubated at 37C for 1 hour as standard pretreatment of the E. faecalis cells.
After 1 hour, samples of 100 L were removed from each group,
serially diluted, plated on tryptone soy agar plates, and incubated at
37C for 2 days. Colony-forming units were counted and recorded as the
baseline viable bacterial numbers.
Challenge Conditions
Immediately after pretreatment, 1 mL of ac-PBS was added to
group 1 and 1 mL of collagen solution to group 2, with the purpose of
making the collagen concentration and the pH of the medium identical
in both groups. The study design also sought to make the possible
disinfectant-neutralizing effect of collagen similar for both groups. After
these additions, the pH of the mixture in both groups was 4.70. Subsequently, 7 mL of calcium hydroxide solution (1.25 mL of saturated
solution of calcium hydroxide diluted with 5.75 mL of distilled water)
was added to both groups. Samples of 100 L were removed at 6, 12,
and 24 hours; serially diluted; and plated on tryptone soy agar plates.
Colony-forming units (CFUs) were counted after incubation at 37C for
2 days. The experiment was repeated twice. Absolute viable bacterial
numbers were converted to log10 values, and the average of the three
experiments was calculated. Bacterial detection limit of the experiment
was 100 CFU/mL.
Adherence Assay
An adherence assay (biofilm assay) was performed to test whether
bacteria bound the free collagen molecules in the bacteria-collagen
pretreatment mixture. The assay was performed as described previously
(15). Briefly, 96-well microtiter plates (Sarstedt Inc, Newton, NC) were
coated with collagen type I (50 g/mL PBS) overnight at 4C. Wells
were blocked with bovine serum albumin (Sigma Chemical Co, 100
g/mL PBS) in order to prevent nonspecific binding of the bacteria to
JOE Volume 35, Number 1, January 2009

plastic surfaces. Bacteria were grown at 46C, washed, and standardized as described previously. The bacteria were pretreated 1 hour either
with collagen or ac-PBS at the same concentrations to which they were
exposed during the main experiment. Aliquots of 200 L of the bacterial
suspensions were then added to the collagen-coated wells and incubated for 2 hours. Nonadherent bacteria were removed by washing with
PBS three times. The remaining bacteria were stained with crystal violet
(1%), and the stain captured by the cells was solubilized by the addition
of ethanol:acetone (4:1). The intensity of the stain was measured spectrophotometrically at OD570 and served as a measure of adhesion to the
well walls. The assay was performed in two parallels and repeated three
times.
The relationship of the OD measurements of the dissolved crystal
violet to bacterial numbers was assessed in a separate methodologic
experiment; suspensions of known bacterial concentrations were dried
onto the sides of wells before the staining procedure. The ensuing OD
readings were used to construct a standard curve for OD versus bacterial cell concentrations. A near-linear relationship was found for a loglog plot of the results (Fig. 1), which was used to transform the OD
measurements to bacterial cell concentrations.

Statistical Analysis
Data obtained were analyzed statistically using a Student t test at
p 0.01 as the level for statistical significance.

Results
The results of the susceptibility experiment are shown in Figure 2.
Baseline viable bacterial numbers were found to be [mean CFU/mL
(standard deviation)]: 7.79 108 (6.0 107) for group 1 and 6.08
108 (1.19 108) for group 2. These mean values corresponded to
log10 values of 8.89 and 8.78, respectively. The baseline bacterial numbers were fewer than the nonpretreated standard yields of OD540
1.0 (1 2 109 CFU/mL), suggesting that some of the bacteria could
not survive the acidity in the pretreatment medium. However, no significant difference was found for the baseline viable numbers of bacteria
between the two experimental groups. There was no significant reduction in bacterial viability at 6 hours, and no significant difference was
found between the groups at this period. However, the number of viable
bacteria decreased significantly in both groups at 12 hours, and significantly more viable bacteria were recovered in group 1 compared with
group 2 (more than 10 times, p 0.01). No bacteria could be cultivated in the 24-hour samples of either group.
The standard curve obtained by matching known numbers of bacteria (the y-axis) and their corresponding OD readings after crystal

Figure 2. The survival of collagen-pretreated and ac-PBSpretreated (collagenadded) bacteria after calcium hydroxide challenge. Average and standard deviations of four experiments. The standard deviations were so small (all less
than 0.1) that the error bars cannot be discerned in the figure.

Resistance of Collagen-associated Planktonic Cells of E. faecalis to Ca(OH)2

Downloaded from ClinicalKey.com at N K P Salve Institute Of Medical Sciences and Research Centre July 19, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.

47

Basic ResearchBiology

Figure 3. The percent adhesion of 46C-grown bacteria to collagen-coated

wells after pretreatment with or without collagen. Average and standard deviations of four experiments.

violet destaining (the x-axis) (Fig. 1) were used to calculate the amount
of the bacteria adhering to the collagen-coated wells. According to these
calculations, collagen-pretreated bacteria were found to adhere significantly less than nonpretreated bacteria to collagen-coated surfaces
(p 0.01, Fig. 3).

Discussion
The main finding of this study was that cells of E. faecalis pretreated with collagen were less susceptible to calcium hydroxide at one
period than those that were not pretreated with collagen.
E. faecalis A197A has been shown to adhere to collagen at increased numbers after growth at 46C (15). Therefore, 46C was chosen as the growth temperature in this study to achieve the collagenadherent phenotype and to test the effect of collagen association by the
bacterium on susceptibility to calcium hydroxide. After growth, potential collagen-adhering bacteria were exposed to free collagen in the
pretreatment medium. The results of the adherence assay suggested that
the free collagen was bound by the bacteria in this step; because the
bacterial cell surfaces were covered by the free collagen, these bacteria
could not further adhere to the immobilized collagen on the wells.
The effect of bacterial adherence to various surfaces on susceptibility to antimicrobial substances has been investigated in previous studies. For example, adhesion by Pseudomonas aeruginosa, Escherichia
coli, and Candida albicans to organic or inorganic surfaces has been
found to increase the resistance of these microorganisms against antimicrobials or adverse conditions through stimulation of biofilm formation, upregulation of efflux pump genes, or production of stress proteins
(18 20). Although these experiments tested microorganisms attached
to a surface, the study presented here examined bacteria at a planktonic
state in which collagen-associated E. faecalis cells exposed to calcium
hydroxide were in suspension. The resistance mechanism of the suspended bacteria may be different from those adhering to a surface. One
example is the difference between the biofilm and the planktonic bacteria. Biofilm bacteria can become up to 1,000 times stronger against an
antimicrobial than their planktonic counterparts by using multiple resistance mechanisms including production of an exopolysaccharide
protective matrix and the modulation of the gene expression pattern
(21). The exact mechanism of the resistance found in this study for the
collagen-associated bacteria is unknown. However, because the bacteria were free floating, the whole surface of the bacterial cell was available for collagen binding. A cover of collagen around the cell, whether
a result of a specific adhesin-ligand binding or a nonspecific physical
coating, may have protected the bacteria against the disinfectant. Although such protection may occur in laboratory conditions, it is not
known whether this may occur in vivo.
48

Kayaoglu et al.

There are also studies that have examined the consequences of E.

faecalis adherence to dentine. In one of these studies, the susceptibility
to calcium hydroxide of cells of E. faecalis that were either allowed to
adhere to dentinal surfaces for 24 hours or prepared as planktonic
suspensions was compared; cell recovery was found to be remarkably
greater for the adhering bacteria at 10- and 100-minute challenge periods as assessed by culture (13). In a recent study, similar in design to
the study presented here, we investigated whether Ace-mediated binding
to collagen conferred resistance on E. faecalis to common endodontic
disinfectants (14). It was found that collagen-associated bacteria were
more resistant against chlorhexidine and iodine-potassium iodide but
were surprisingly more susceptible to calcium hydroxide compared
with bacteria not exposed to collagen (14). This finding and the results
of the study presented here regarding the susceptibility of collagenassociated cells to calcium hydroxide are in contrast. Although the
experimental conditions were quite similar in both studies, there were,
however, some differences; in the first study, the bacteria were challenged with a higher concentration of calcium hydroxide solution and
for a relatively short duration (6 hours), whereas, in the study presented
here, we used a lower concentration but challenged the bacteria for a
longer time (24 hours). At this timeframe, besides the antibacterial
effect of the calcium hydroxide, it is also possible that starvation of the
bacteria because of a lack of nutrients in the challenge medium could
have affected the cultivability of the bacteria. Another difference was the
bacterial strains tested. In the presented study, we tested E. faecalis
A197A. This strain was isolated from the root canal of a tooth with
persistent endodontic infection (17). Calcium hydroxide was the most
commonly used medicament in the study in which this strain was obtained (17). Therefore, it is possible that the A197A strain was a survivor
of calcium hydroxide. In the previous study, we tested E. faecalis OG1RF
(14). This strain was derived from E. faecalis OG1 by selection for
resistance to rifampin and fusidic acid (22, 23), and OG1 (formerly
called 2SaR) was originally isolated from human saliva (24). The
OG1RF strain has thus become a laboratory strain and has been used as
a reference strain also in other studies (7, 8, 25). It has been suggested
that bacterial genomes evolve during serial in vitro passage in laboratory conditions and important genomic and phenotypic differences
emerge between laboratory reference strains and clinical isolates of the
same species (26). The difference between the collagen-associated
OG1RF and A197A for resistance against calcium hydroxide could also
be caused by this.
The results of this study, performed by using an endodontic isolate
of E. faecalis, suggested that collagen association at a planktonic state
increased the bacterial resistance to calcium hydroxide. Future studies
designed to test the susceptibility of E. faecalis adhering to immobilized
collagen will provide complementary information to the understanding
of the antimicrobial resistance mechanisms of this species.

References
1. Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev 1990;
3:46 65.
2. Siqueira JF Jr, Ras IN. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 2004;97:8594.
3. Sedgley C, Nagel A, Dahln G, Reit C, Molander A. Real-time quantitative polymerase
chain reaction and culture analyses of Enterococcus faecalis in root canals. J Endod
2006;32:1737.
4. Gomes BP, Pinheiro ET, Jacinto RC, Zaia AA, Ferraz CC, Souza-Filho FJ. Microbial
analysis of canals of root-filled teeth with periapical lesions using polymerase chain
reaction. J Endod 2008;34:537 40.
5. Love RM. Enterococcus faecalisa mechanism for its role in endodontic failure.
Int Endod J 2001;34:399 405.
6. Rich RL, Kreikemeyer B, Owens RT, et al. Ace is a collagen-binding MSCRAMM from
Enterococcus faecalis. J Biol Chem 1999;274:26939 45.

JOE Volume 35, Number 1, January 2009

Downloaded from ClinicalKey.com at N K P Salve Institute Of Medical Sciences and Research Centre July 19, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.

Basic ResearchBiology
7. Nallapareddy SR, Singh KV, Duh RW, Weinstock GM, Murray BE. Diversity of ace, a
gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of
ace during human infections. Infect Immun 2000;68:5210 7.
8. Kowalski WJ, Kasper EL, Hatton JF, Murray BE, Nallapareddy SR, Gillespie MJ. Enterococcus faecalis adhesin, Ace, mediates attachment to particulate dentin. J Endod
2006;32:634 7.
9. Estrela C, Rodrigues de Araujo Estrela C, Bammann LL, Pecora JD. Two methods to
evaluate the antimicrobial action of calcium hydroxide paste. J Endod 2001;27:720 3.
10. Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro
antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J
2001;34:424 8.
11. rstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of
experimentally infected dentinal tubules. Endod Dent Traumatol 1990;6:1429.
12. Haapasalo HK, Siren EK, Waltimo TM, rstavik D, Haapasalo MP. Inactivation of local
root canal medicaments by dentine: an in vitro study. Int Endod J 2000;33:126 31.
13. Brndle N, Zehnder M, Weiger R, Waltimo T. Impact of growth conditions on susceptibility of five microbial species to alkaline stress. J Endod 2008;34:579 82.
14. Kayaoglu G, Erten H, rstavik D. Possible role of the adhesin ace and collagen
adherence in conveying resistance to disinfectants on Enterococcus faecalis. Oral
Microbiol Immunol 2008;23:449 54.
15. Kayaoglu G, Erten H, rstavik D. Growth at high pH increases Enterococcus faecalis
adhesion to collagen. Int Endod J 2005;38:389 96.
16. Reynaud af Geijersstam A. Genetic variations and comparative analysis of virulence
determinants in Enterococcus isolates originating from the root canal (dissertation).
Oslo: University of Oslo, 2006.

JOE Volume 35, Number 1, January 2009

17. Siren EK, Haapasalo MP, Ranta K, Salmi P, Kerosuo EN. Microbiological findings and
clinical treatment procedures in endodontic cases selected for microbiological investigation. Int Endod J 1997;30:915.
18. Trafny EA, Kowalska K, Grzybowski J. Adhesion of Pseudomonas aeruginosa to
collagen biomaterials: effect of amikacin and ciprofloxacin on the colonization and
survival of the adherent organisms. J Biomed Mater Res 1998;41:5939.
19. Mateus C, Crow SA, Ahearn DG. Adherence of Candida albicans to silicone induces
immediate enhanced tolerance to fluconazole. Antimicrob. Agents Chemother
2004;48:3358 66.
20. Dahan S, Knutton S, Shaw RK, Crepin VF, Dougan G, Frankel G. Transcriptome of
enterohemorrhagic Escherichia coli O157 adhering to eukaryotic plasma membranes. Infect Immun 2004;72:54529.
21. Mah TF, OToole GA. Mechanisms of biofilm resistance to antimicrobial agents.
Trends Microbiol 2001;9:34 9.
22. Dunny GM, Brown BL, Clewell DB. Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone. Proc Natl Acad Sci U S A
1978;75:3479 83.
23. Murray BE, Singh KV, Ross RP, Heath JD, Dunny GM, Weinstock GM. Generation of
restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J Bacteriol 1993;175:5216 23.
24. Gold OG, Jordan HV, van Houte J. The prevalence of enterococci in the human mouth
and their pathogenicity in animal models. Arch Oral Biol 1975;20:4737.
25. Nallapareddy SR, Duh RW, Singh KV, Murray BE. Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and
pulsed-field gel electrophoresis. J Clin Microbiol 2002;40:868 76.
26. Fux CA, Shirtliff M, Stoodley P, Costerton JW. Can laboratory reference strains mirror
real-world pathogenesis? Trends Microbiol 2005;13:58 63.

Resistance of Collagen-associated Planktonic Cells of E. faecalis to Ca(OH)2

Downloaded from ClinicalKey.com at N K P Salve Institute Of Medical Sciences and Research Centre July 19, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.

49