ARTÍCULOS ORIGINALES
Enterococcus faecalis biofilm. Formation and development in vitro observed by scanning electron microscopy
María de los Á. Bulacio1, Lucas R. Galván1, Cristina Gaudioso2, Rosa Cangemi2, Marta I. Erimbaue1
1 Department of Endodontics, School of Dentistry, National University of Tucumán, Tucumán, Argentina..
2 Department of Microbiology, School of Biochemistry, National University of Tucumán, Tucumán, Argentina.
CORRESPONDENCE Dr.Maria de los Angeles Bulacio. Crisostomo Alvarez 1487. CP 4000 San Miguel de Tucuman, Tucuman. Argentina maritabulacio@hotmail.com
ABSTRACT
Biofilm produced by Enterococcus faecalis isolated from root canals was detected by growing it on microplates and using 10% crystal violet stain, elution with alcohol and three procedures: no fixation, heat fixation and 10% formaldehyde fixation. The biofilm was evaluated using a Versamax Microplate Reader (USA). Twenty sterile root portions were incubated in TS broth with E. faecalis (108) for 48 hours, 4, 7, 14 and 30 days, after which they were processed and observed by scanning electron microscopy (SEM). Significantly more biofilm was found on the microplates for formaldehyde fixation than for heat fixation or no fixation (ANOVA p<0.0001). SEM showed E. faecalis growth at all times and biofilm development as from 14 days’ incubation. Fixation with 10% formaldehyde was the most appropriate technique for detecting E. faecalis biofilm development on microplates. SEM confirmed biofilm formation after 14 days incubation.
Key words: Biofilm; Enterococcus faecalis; Scanning electron microscopy.
RESUMEN
Biofilm de Enterococcus faecalis. Producción in vitro y desarrollo observado con microscopía electrónica de barrido
Se realizo la deteccion de biofilm de Enterococcus faecalis aislados de conductos radiculares, desarro llandolos en microcubetas realizando tincion con Cristal Violeta 10 %, elucion con alcohol y tres procedimientos: sin fijar, fijacion con calor y fija cion con formaldehido 10%. Se evaluo el biofilm formado realizando la lectura con lector de microplaca Versamax-Microplate-Reader (USA). Se incubaron 20 porciones radiculares esteriles en el medio TS caldo con E. faecalis (108) durante 48 hs, 4, 7, 14 y 30 dias. Transcurrido cada tiempo experi mental se procesaron y observaron al microscopio electronico de barrido (MEB). Se evidencio significativamente mayor formacion de biofilm en microplacas cuando se realizo fijacion con formaldehido, que al fijar con calor y sin fijar (ANOVA p<0,0001). Al MEB se observo crecimiento del E. faecalis en todos los tiempos y desarrollo de biofilm a partir de 14 dias de incubacion. La fijacion con formaldehido 10% fue la tecnica mas apropiada para detectar biofilm de E. faecalis desarrollado en microplacas. Mediante el MEB se confirmo la formacion de biofilm despues de 14 dias de incubacion.
Palabras clave: Biofilm; Enterococos faecalis; Microscopia electronica de barrido.
INTRODUCTION
Enterococci are natural inhabitants of the oral cavity
and normal intestinal flora, and are recognized as
opportunistic pathogens. Enterococcus faecalis is
the most common species, and is responsible for 80
- 90% of human infections1.
Research has shown that areas in the canal which are
inaccessible to instruments and irrigants may remain
contaminated after biomechanical preparation and
be responsible for persistent infection2.
Cases of apical periodontitis resistant to conventional
treatment3 are attributed to the presence of
biofilm inside the canal and at the apical end of
teeth with pulp necrosis and chronic periapicial
lesion4.
SEM observations of E. faecalis have shown that it
adheres to collagen structures, colonizes dentin
surfaces and may progress towards dentin tubules
and form mature biofilm5.
The aim of this study was to compare detection on
microplates of biofilm formed by Enterococcus
faecalis isolated from root canals, in Tryptone Soya
(TS) broth supplemented with 1% glucose and 10%
human serum, using three different techniques: no fixation, heat fixation and 10% formaldehyde
fixation, and observing the development of biofilm
on root portions under scanning electron microscopy
(SEM) at 48 hours, 4, 7, 14 and 30 days.
MATERIALS AND METHODS
The study included 10 patients who attend the
Endodontics Department at the School of Dentistry
at the National University of Tucuman and provided
informed consent, with indication for retreatment
of teeth due to the persistence of chronic-type
periapical lesion.
After clinical-radiographic diagnosis, the patients
rinsed their mouths with 0.12% chlorhexidine
solution (Plac Out. Microsules Bernabo, Argentina).
The area was completely isolated and the operative
field disinfected with 1% chlorhexidine solution.
Caries was eliminated using hand instruments and
irrigation with 1% chlorhexidine. The access cavity
was prepared with water-cooled high-speed
diamond burs. The operative field was disinfected
and the canal filling material was removed with Hfiles,
followed by irrigation with 1 mL distilled
water. Samples were taken with two standard N° 20
paper points placed inside the root canal for one
minute, and then placed individually in test tubes
containing TS broth and transferred immediately to
the laboratory.
In the laboratory we performed a semi-quantitative
study by dilution in the following media: Azide
agar for Enterococcus and Staphylococcus,
MacConkey agar for Gram-negative bacilli, Blood
agar for Streptococcus. The different colonies were
counted and E. faecalis was identified according to
morphological and physiological features6.
The three strains with greatest development on
plates were selected and their ability to produce
biofilm was studied.
Biofilm formation on microplates
The culture medium used was Trypticase Soya (TS)
broth supplemented with 1% glucose and 10%
human serum. Three E. faecalis strains isolated
from the canals were selected and called strains 1,
2 and 3.
Microplates were loaded with 200 μl. of the
medium with inoculum at a concentration of 108,
incubated at 37°C for 24 hours, without stirring, in
triplicate. To detect biofilm production, the
microplates were stained with 10% crystal violet
(C.V.), washed with 95% ethanol to remove excess
dye and subject to one of three different procedures:
• Without fixation
• Heat fixation
• Formaldehyde fixation
The biofilm formed was evaluated by measuring absorbance using a Versamax Microplate Reader (USA).
Biofilm development observed with scanning
electron microscope (SEM)
Twenty disks were cut from the middle third of the
roots of recently extracted upper central incisors
using a water-cooled, high-speed N° 1090
cylindrical bur (Sorensen, Brazil). The disks were
sterilized and placed individually in TS broth
enriched with 1% glucose and 10% human serum,
and inoculated with E. faecalis at a concentration
of 108. The media were refreshed every 72 hours
with the same inoculum concentration. Five
experimental incubation times were used: 48 hs, 4,
7, 14 and 30 days, after which the samples were
removed from the medium and placed individually
in a solution of 8% paraformaldehyde, phosphate
buffer and 16% glutaraldehyde and fixed for
48 hours at 4°C. They were washed 3 times for
10 minutes with phosphate buffer. A 1/1 osmium/
phosphate buffer solution (2% OsO4) was added
and left overnight. The osmium was discarded and
the samples were washed twice for 10 minutes with
30% ethanol. The samples were dehydrated by
passing through a series of alcohol concentrations:
ethanol 50%, 70%, 80%, 90%, and 3 times 100%,
with 10 minutes for each step. The 100% ethanol
was discarded and 100% acetone added for 1 hour7.
The samples were mounted on an aluminum
specimen holder, sputter-coated with gold and
viewed under high vacuum with a JEOL JSM-35CF
microscope (Tokyo, Japan). Microphotographs
were taken at 10000 X.
RESULTS
Biofilm forming assay on microplates
Strain 2 had the greatest development in TS broth
supplemented with 1% glucose and 10% human
serum for all three techniques used (Table 1), so we
selected it for the SEM study. The control value
(0.0360) was taken from the reading for the
microplate without biofilm.
The values obtained with strain No. 2 for the
formaldehyde fixation treatment were significantly
higher than for the other treatments (ANOVA,
p < 0.0001), Fig. 1.
Table 1: Analysis of Variance. Absorbance reading on biofilms in nm for three Enterococcus faecalis strains
using three techniques. The mean is the average of the readings for the four wells seeded with the
same strain.
Fig. 1: Biofilm obtained. Absorbance (nm) measurements of E.
faecalis strain 2, with three different fixation procedures.
Biofilm development observed with scanning
electron microscope
Figures 2 to 6 show the development of Enterococcus
faecalis under SEM at the different times. Biofilm is
apparent as from 14 days.
Fig. 2: SEM. E. faecalis development at 48 hs. The reference
line corresponds to 1 μm.
Fig. 3: SEM. E. faecalis development at 4 days. The reference
line corresponds to 1 μm
Fig. 4: SEM. E. faecalis development at 7 days. The reference
line corresponds to 1 μm.
Fig. 5: SEM. E. faecalis biofilm at 14 days. The reference line
corresponds to 1 μm.
Fig. 6: SEM. Mature E. faecalis biofilm at 30 days. The reference
line corresponds to 1 μm.
DISCUSSION
This study used the ability of E. faecalis to form
biofilm on polystyrene6, which occurs due to the
presence of a surface protein, the Esp-encoding gene (esp)8, although Kristich et al.9 claim that E. faecalis is
able to form biofilms in absence of this gene.
Biofilm production often depends on factors such
as cell density and glucose concentration10, with
several authors having proved that glucose
supplement increases biofilm formation by
E. faecalis9-11 and other papers reporting that
biofilms are denser in presence of glucose12. Our
study took this factor into account.
Not all strains have the same biofilm-producing
capacity. Our study used 3 E. faecalis strains
isolated from root canals with endodontic failure.
We selected the strain with the greatest capacity to
produce biofilm on microplates, and used it to
contaminate root dentin and observe biofilm
development under SEM.
Duggan and Sedgley13 found no statistically
significant difference in the formation of biofilm by
E. faecalis obtained from root canals, oral cavity
and other parts of the body, using a similar culture
medium and evaluating the optical density of the
microplates, suggesting that the origin of the strain
did not influence the result of their study.
Zheng et al.14 found that 75.47% of the E. faecalis
strains isolated from root canals with endodontic
failure were able to develop biofilm.
Wang et al.15 also found high biofilm forming rates by
E. faecalis isolated from root canals with endodontic
failure, employing growth on microplates and SEM
observations.
Wilcox16 report that early formaldehyde fixation may
help show biofilm formation, and this technique was
employed successfully in our study.
Our study found that E. faecalis biofilm was
observable at 14 days and mature biofilm at 30 days.
Guerreiro-Tanomaru et al.17 report similar results.
They used different substrates such as bovine dentin
and bone, hydroxyapatite and gutta-percha, and
reported biofilm development at 14 and 21 days,
which they observed by using staining methods
and confocal microscopy, finding the greatest
development on hydroxyapatite. Wu et al.18 inoculated E. faecalis on human teeth for 4 weeks to
obtain biolfilm. Somayaji et al.19 used SEM and found
a high percentage of biofilm development 45 days.
Du et al.20 made observations under confocal
microscope on pieces of dentin incubated with E.
faecalis for 1 and 21 days, finding young and a mature
biofilm, respectively, at those times. Our study shows that under the experimental conditions applied, it is
possible to generate mature E. faecalis biofilm at 30
days’ incubation on the surface of human root dentin.
CONCLUSIONS
Enterococcus faecalis isolated from root canals
was able to produce biofilm on microplates,
which was best seen using 10% crystal violet
stain, elution with alcohol and fixation with 10%
formaldehyde.
Mature E. faecalis biofilm formation was confirmed
by SEM after 30 days’ incubation, using pieces of
root dentin in TS broth supplemented with 10%
human serum and 1% glucose.
ACKNOWLEDGMENTS
Partially supported by CIUNT,Tucuman,Argentina. We thank Dr. Marcela D’Urso for the statistical analysis.
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