Received Date : 22-May-2013

Accepted Article Revised Date : 07-Apr-2014

Accepted Date : 16-Apr-2014

Article type : Original Scientific Article

Clonal diversity in biofilm formation by Enterococcus faecalis in response to environmental

stress associated with endodontic irrigants and medicaments.

C.E. Wilson1, P.C. Cathro2, A.H. Rogers1, N. Briggs3, P.S. Zilm1

1
Oral Microbiology, School of Dentistry. 2Discipline of Endodontics, School of Dentistry, 3Data

Management and Analysis Centre, The University of Adelaide. Adelaide, Australia.

Running Title: Genetic diversity in E. faecalis biofilm formation

Keywords: Enterococcus faecalis, Endodontics, biofilm, stress response.

Corresponding author.

Dr. Peter Zilm.

Oral Microbiology Laboratory, School of Dentistry, The University of Adelaide, North Terrace,

Adelaide, South Australia, 5005 Australia.

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
'Accepted Article', doi: 10.1111/iej.12301

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 Ph. 61 8 83135676
Accepted Article
Fax 61 8 83133444

Email peter.zilm@adelaide.edu.au

ABSTRACT

Aim To determine whether clonal diversity within E. faecalis affects biofilm formation when exposed

to antimicrobial compounds found in endodontic medicaments and irrigants.

Methodology Five human isolates of E. faecalis were compared; biofilms were grown in microtitre

trays in the presence of sodium hypochlorite, calcium hydroxide, chlorhexidine, tetracycline or

clindamycin. Biofilms were quantified by staining with crystal violet and optical density determined

with a microplate reader. Slime production (an amorphous extracellular matrix comprising

polysaccharides, glycoproteins and glycolipids loosely attached to the cell surface) was determined

qualitatively by growth on Congo red agar plates. Linear mixed models were used to examine whether

medicaments affected biofilm growth of the isolates in the presence of the medicaments or irrigants.

Results Overall, different endodontic antimicrobials significantly altered biofilm growth in E. faecalis

isolates. Two E. faecalis isolates significantly (p < 0.0001) increased biofilm formation in the presence

of tetracycline and one in the presence of NaOCl (p = 0.018). Qualitatively, slime production also

varied between isolates and correlated with biofilm production.

Conclusions: When subjected to sub-MIC levels of antimicrobial compounds found in endodontic

medicaments, E. faecalis isolates demonstrated significant clonal variation in their capacity to form

biofilms. Interestingly, there was a correlation between slime production and the ability of isolates to

form a biofilm in the presence of antimicrobials. The results indicate that isolates of E. faecalis which

form biofilms in response to endodontic medicaments may be more likely to survive endodontic

treatment.

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 INTRODUCTION
Accepted Article
Enterococcus faecalis is an important species amongst the microbial survivors of root canal treatment

(Sundqvist et al. 1998). In most instances they are harmless commensal organisms (Kayaoglu &

Ørstavik 2004) but, in certain circumstances they can become opportunistic pathogens (Moellering

1992). Endodontic irrigants and medicaments have commonly been used to remove bacteria from the

root canal system (Sjögren et al. 1991). However, certain bacterial species have shown resistance to

some of these antimicrobial agents (Gomes et al. 2001, El Karim et al. 2007). While the prevalence of

E. faecalis in primary endodontic infections is low, they become prominent in persistent endodontic

infections (Rôças et al. 2004, Siqueira & Rôças 2004, Stuart et al. 2006) and with the development of

multiple drug resistant strains, E. faecalis has also been identified as a significant nosocomial

pathogen (Huycke et al. 1998).

The ability to form a biofilm is a significant factor contributing to the persistence of E. faecalis during

root canal treatment (Jenkinson & Lappin-Scott 2001). Biofilms are produced by aggregations of

bacteria on hydrated surfaces forming microbial communities enclosed in an abundant extracellular

polymeric matrix (Hall-Stoodley & Costerton 2004). Bacteria that exist in biofilms have been shown

to have greater resistance to antimicrobial agents (Donlan & Costerton 2002, Abdullah et al. 2005).

Also, several virulence factors enhance the ability of E. faecalis to produce biofilms (Ciardi et al.

1977, Waar et al. 2002).

Slime is composed of an amorphous glycocalyx which is loosely attached to the cell surface and forms

a covering that aids in adherence to surfaces (Christensen et al. 1982). It is thought to provide

protection against host defences and antimicrobials (Farber et al. 1990, Barrio et al. 2000). In

particular, slime production has been linked to the ability of bacteria to resist the effects of various

antibiotic classes (Ciftci et al. 2009).

Therefore, differences in the ability of strains of E. faecalis to produce slime could influence their

survival during endodontic treatment.

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 Clonal diversity amongst E. faecalis isolates has enabled the species to successfully colonise a range
Accepted Article of locations in the human body as well as other animal and environmental niches (Jett et al. 1994,

Fisher & Phillips 2009). Sequencing of the genome of E. faecalis V583 has revealed that it contains a

core of stable essential genes accompanied by exogenously acquired DNA (Paulsen et al. 2003, Aakra

et al. 2007). Examination of the acquired DNA has shown that it is comprised of genes involved with

production of virulence proteins, including those involved in biofilm formation (Aakra et al. 2007).

One such protein is the surface adhesin, “aggregation substance”, which improves bacterial binding to

the collagen component of dentine (Kayaoglu & Ørstavik 2004). It also encourages positive synergy

between biofilm members, whereby one adhering microorganism stimulates the adherence of others

(Waar et al. 2002). Therefore, differences in possession of virulence factors amongst members of the

species could give them an advantage in their ability to persist in the root canal environment.

Thus far, the origin of E. faecalis implicated in persistent endodontic infections is yet to be

established, with both endogenous and exogenous sources postulated (Zehnder & Guggenheim 2009,

Vidana et al. 2010). Vidana et al. (2010) compared E. faecalis isolates from persistent endodontic

infections with isolates from the saliva and faeces of the same patients. They found that the patient’s

endogenous E. faecalis was not genotypically the same as the root canal isolates and, therefore,

concluded that an exogenous source of the bacteria was more likely. Zehnder & Guggenheim (2009)

hypothesised that if E. faecalis were a transitory oral species then food would be the most likely

source. With no confirmed source of E. faecalis causing infection, isolates from a range of possible

origins were used in the present study.

Commonly used endodontic irrigants and medicaments create environmental stress during growth of

bacteria. Sodium hypochlorite (NaOCl) is widely used as a root canal irrigant due to its antimicrobial

and tissue dissolving capabilities (Clarkson & Moule 1998). Furthermore, the formation of chlorine, a

strong oxidant that combines with amino groups to produce chloramines, interferes with enzyme

activity (Estrela et al. 2002). NaOCl also has low viscosity, which enables it to penetrate dentinal

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 tubules and flush away debris (Zou et al. 2010). Chlorhexidine (CHx) has antimicrobial activity
Accepted Article against some bacteria and shows substantivity within the root canal (Önçağ et al. 2003). Its principal

antimicrobial property is linked to its capacity to cause membrane disruption (Kuyyakond & Quesnel

1992). Calcium hydroxide creates alkaline conditions that interfere with enzymatic reactions and

damage cytoplasmic membranes (Athanassiadis et al. 2007). The antibiotics contained in Ledermix®

(tetracycline) and Odontopast® (clindamycin) appear to have only a limited antibacterial effect against

endodontic pathogens (Athanassiadis et al. 2009, 2010) and they are principally used for their

corticosteroid component, which reduces the pain associated with acute endodontic infections (El

Karim et al. 2007).

Clinically, differences in the virulence of E. faecalis may explain, in some cases, the persistence of E.

faecalis following optimal treatment. Although several studies have confirmed the presence of genetic

diversity amongst isolates of E. faecalis (Sedgley et al. 2004, McBride et al. 2007, Zoletti et al. 2011),

there is little information on whether different isolates of E. faecalis vary in their ability to grow as a

biofilm as part of their response to a stressful environment (Duggan & Sedgley 2007).

The aim of the present study was to compare biofilm formation between E. faecalis isolates when

subjected to environmental stress caused by the presence of endodontic medicaments or irrigants. The

study also aimed to determine whether differences in slime production existed between the isolates

and whether this was associated with biofilm growth. The null hypothesis therefore was that the

presence of endodontic medicaments or irrigants would not increase biofilm formation by E. faecalis

and that biofilm formation was not correlated to slime production.

MATERIALS AND METHODS

Bacterial isolates and culture conditions

Three clinical isolates of E. faecalis were obtained from Professor Ivan Bastian (Microbiology

Laboratory, SA Pathology, Adelaide, Australia). They included a skin isolate from a proximal right

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 toe (3325), a urine isolate (3326) and a metatarsal bone isolate (3240). An oral isolate (JKD 15036)
Accepted Article was generously donated by A/Prof Stuart Dashper (School of Dentistry, Melbourne, University of

Melbourne, Australia) and a blood isolate (ATCC 700802, V583) was purchased from the ATCC

(Cryosite, Sydney, Australia). Cultures were maintained on blood agar (Oxoid, Melbourne, Australia)

and purity was periodically checked by plating onto Bile Aesculin agar (Oxoid). To prepare the

inoculum for the biofilm assay, isolates were transferred from blood agar plates to Todd Hewitt broth

(THB) (Oxoid) and incubated overnight at 37°C. After incubation, the OD595nm values were

normalized to 0.4. The inoculation culture was then added to a mictotitre tray (Becton Dickinson,

Franklin Lakes, NJ, USA) in a ratio of 1(inoculum):40 with either THB or THB mixed with an

antimicrobial agent to a final volume of 200μL/well.

Biofilm Assay

The method used for the biofilm assay was modified from that described by Toledo-Arana et al.

(2001). Growth and biofilm formation were allowed to occur over 48 hours at 37°C. Planktonic

bacteria were then removed and the microtitre trays washed 3 times with 0.9% saline to ensure that

only the attached biofilm remained. Plates were left to dry at 25oC for 1 hour and the biofilms stained

with 200µL of 1% crystal violet (Oxoid) for 15 minutes. The crystal violet was then carefully removed

and the trays washed 3 times with 0.9% saline. Trays were allowed to dry for 1 hour before

solubilising the adhered crystal violet with 200µL ethanol:acetone (80:20 vol/vol). The microtitre

trays were left on a plate shaker (Ratek Instruments, Melbourne, Australia) for 30 minutes to ensure

homogeneous mixing of the well contents. Optical density of the wells was read at 595nm using a

microplate reader (Biotek, Winooski, VT. USA). The contents from a single well from each row were

plated onto Bile aesculin agar to ensure that no contamination had occurred. Each biofilm assay had a

total of 36 replicates (3 rows of a mictrotitre tray) for each isolate.

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 Antibiotic Sensitivity
Accepted Article
As E. faecalis is resistant to many antibiotics, the sensitivity of the isolates to antibiotics present in

endodontic medicaments was tested. A well-plate method was used to determine sensitivity to

Odontopaste® (Aust. Dental Manufacturing, Brisbane, Australia) and Ledermix® (Sigma

Pharmaceuticals, Melbourne. Australia), which contain the antibiotics clindamycin and tetracycline,

respectively. Overnight cultures of the E. faecalis isolates were spread over blood agar plates and a

sterile tube of 5 mm diameter was used to cut wells in the agar plates. The endodontic medicaments

(50 µL) were introduced into the wells and the plates incubated at 37ºC for 48 hours. Each

medicament was tested in triplicate for all isolates. A zone of growth inhibition occurring around the

medicaments revealed sensitive isolates. Only isolates sensitive to the medicaments were used to

determine the minimum inhibitory concentration. For resistant isolates, both the medicament and the

isolate were excluded from the biofilm assay.

Minimum Inhibitory Concentration (MIC)

Serial dilutions of endodontic antimicrobials were used to determine the MIC so that biofilm

formation by E. faecalis isolates could be quantified under stressful growth conditions (i.e. the dilution

below the MIC). The antimicrobials used were: 0.95% w/w NaOCl (Milton Antibacterial Solution,

Sydney, Australia); 5% w/v CHx (Johnson & Johnson, Sydney Australia); Ca(OH)2 (Ajax chemical,

Melbourne, Australia); tetracycline (Sigma); and clindamycin (Sigma) and the appropriate

concentration of each was determined as follows:

Each chemical was added to separate tubes containing THB at final concentrations of 0.475% v/v

NaOCl; 2.5% v/v CHx; 20% w/v Ca(OH)2; 12.5% w/v tetracycline; 0.25% w/v clindamycin and

200µL were dispensed into the first well of each row of the microtitre tray. The remaining wells in

each row contained 100µL THB. Aliquots of 100µL were used to make serial dilutions. The inoculum

containing each isolate (10µL) was then added to each well and incubated for 24 hours at 37o C. After

incubation, 100µL of culture was removed from each well and transferred to a microtitre tray so that

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 bacterial (planktonic) growth (OD595nm) could be assessed using a microplate reader (Biotek). Each
Accepted Article MIC was done in triplicate (3 rows of a mictrotitre tray) for each isolate. The concentration of the

antimicrobial at the dilution below the MIC was chosen as the final concentration for the biofilm

assay.

Biofilm Assay in the Presence of Endodontic Antimicrobials

The bacterial inoculum was diluted 1:40 with THB containing each antimicrobial at a concentration

just below the MIC. Biofilms were grown over 48 hours and quantified as described previously. Each

assay was done in triplicate (3 rows of a mictrotitre tray) for each isolate. Control samples contained

the antimicrobial but were un-inoculated to control for coating of the microtitre tray well surface.

Slime Production

Slime production was determined by culturing the E. faecalis isolates (48 hours at 37ºC) on Congo red

agar plates containing 0.8g/L Congo red and 36g/L saccharose in Brain Heart Infusion agar (Oxoid).

Those isolates positive for slime production formed black colonies whilst isolates not able to produce

slime remained pink. Isolates that were only weak slime producers formed colonies that were partially

black (Kouidhi et al. 2011).

Statistical analysis

The ability of E. faecalis isolates to form a biofilm in the presence or absence of the antimicrobials

was analysed statistically using a linear mixed model allowing for the fixed effects of the isolate, the

irrigant or medicament and their interaction. A random effect of each row to account for the

correlation among replicates was also included. Estimated means and 95% confidence intervals were

obtained for each irrigant and medicament. Differences in biofilm growth between the medicament

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 conditions were tested within each isolate. Raw p-values as well as those adjusted for multiple
Accepted Article comparisons (using Holm’s Stepdown Bonferroni procedure (Holm 1979) were also calculated. The

adjusted p-values resulted in no change to the conclusions from the raw p-values. The individual

conditions of NaOCl, CHx, Ca(OH)2, clindamycin and tetracycline were analysed to determine

whether there was significant variation in the optical density measurements for specific isolates tested.

RESULTS

Differences amongst the isolates were found in their resistance to tetracycline and clindamycin.

Amongst the five isolates tested, three phenotypes were observed: two isolates (3240 and JKD 15036)

were resistant (bacterial growth to the edge of the well) to both Ledermix® and Odontopaste®; 3325

and 3326 were sensitive (zone of no growth around the well) only to Odontopaste®; and V583 was

sensitive only to Ledermix® .

In the presence of NaOCl (Figure 1), significantly (p < 0.0001) lower levels of biofilm production

occurred for isolates V583, JKD 15036, 3325 and 3326 when compared with the control (THB),

whereas for 3240, significantly (p = 0.018) higher levels of biofilm formation occurred.

The presence of CHx (Figure 2) at sub-inhibitory concentrations resulted in significantly (p < 0.001)

decreased biofilm formation for all isolates. Importantly, the degree to which biofilm formation was

reduced varied depending on the isolate tested. The mean plot shown in Figure 2 shows that the

difference in biofilm formation between THB and CHx for the 3240 isolate was smaller than for all

other isolates (although this was not tested statistically).

Comparison of the biofilm formation by E. faecalis isolates when subjected to Ca(OH)2 (Figure 3)

again showed differences in their responses to the presence of the antimicrobial. V583, (p < 0.001)

JKD 15036, (p = 0.020) and 3326 (p = 0.035) isolates all showed significantly lower levels of biofilm

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 formation in the presence of sub-inhibitory concentrations of Ca(OH)2. By contrast, the reduction in
Accepted Article biofilm formation for 3240 and 3325 was not significant compared to controls.

In the presence of sub-inhibitory levels of clindamycin (Figure 4), JKD 15036 (p = 0.004) and 3326 (p

< 0.0001) produced significantly less biofilm and the decrease was greater for the 3326 than JKD

15036. The level of biofilm formation in isolates 3240 and 3325 was not significantly different in the

presence of clindamycin.

None of the isolates (3240, JKD15036, V583) which were sensitive to tetracycline showed a decline in

biofilm formation in the presence of sub-inhibitory levels of tetracycline (Figure 5). JKD 15036

showed no significant difference in biofilm growth. In contrast, 3240 and V583 showed significantly

(p<0.0001) elevated biofilm formation in the presence of tetracycline, with the greatest increase shown

by isolate 3240.

Isolate 3240 was the only strong slime producer, whilst JKD 15036 was the only non-slime producer

present. Isolates 3325, 3326 and V583 were all weak slime producers (Figure 6).

DISCUSSION

The microtitre tray method used to measure biofilm formation between E. faecalis isolates has been

commonly used (Toledo-Arana et al. 2001, Hancock & Perego 2004, Mohamed et al. 2004). For the

present study, a fixed incubation period (48hours) was chosen so that the bacteria had sufficient time

to establish a quantifiable biofilm. Similar time frames have been use in other studies (Niemira &

Solomon 2005, Agarwal et al. 2011).

It has been shown that the E. faecalis genome contains the gene, bopABCD, which enables the bacteria

to attach to plastic surfaces such as the polystyrene surface of a microtitre tray (Hufnagel et al. 2004).

The method enables quantification of total biofilm mass, including both viable and non-viable cells.

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 Although crystal violet dye has been used extensively (Stepanović et al. 2000, Toledo-Arana et al.
Accepted Article 2001, Mohamed et al. 2004), several aspects of the method which impact on the amount of biofilm

quantified are often not adequately outlined (Extremina et al. 2011). The protocols used for the assay

in the present study followed those of Extremina et al. (2011) who identified several important

parameters of the staining protocol that can affect results. Despite the difficulties associated with the

microtitre tray biofilm assay, the ability to incorporate a high number of replicates while maintaining

the same environmental conditions is one of the advantages of the approach.

Clonal diversity amongst the isolates of E. faecalis was demonstrated by their resistance to the

antibiotics clindamycin and tetracycline. Previous studies have shown that E. faecalis isolates are

highly resistant to tetracycline and clindamycin (Aarestrup et al. 2000, Duh et al. 2001). Although

Ledermix® and Odontopaste® are currently used for relief of pain, rather than removal of bacteria, the

identification of isolates sensitive to these medicaments in the present study shows that in some cases,

they may help to reduce the numbers of E. faecalis present.

The selection of a range of E. faecalis isolates was chosen to reflect the many possible sources of E.

faecalis that might cause infection of the root canal system. Historically, E. faecalis was believed to be

a common inhabitant of the oral cavity (Gold et al. 1975). However, more recent studies have shown

evidence to the contrary (Aas et al. 2005). Since E. faecalis is transitory in the oral cavity it is possible

that infection could come from many origins including human, animal and food sources. The potential

for variation amongst the species caused by the acquisition of virulence factors is clinically significant.

Several authors have studied differences in the genetic make-up of E. faecalis. McBride et al. (2007)

used 106 isolates from variable locations to show diversity amongst the species. They found variation

in the capsule locus, resistance to antimicrobials and possession of virulence proteins including

cytolysin and gelatinase. Other studies have confirmed the existence of considerable genetic

polymorphism amongst enterococcal isolates (Sedgley et al. 2004, Aakra et al. 2007, Zoletti et al.

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 2011). Of particular relevance to the present study was the demonstration that E. faecalis is able
Accepted Article persist in the presence of various endodontic irrigants and medicaments (Byström et al. 1985, Krause

et al. 2007).

Clonal diversity amongst E. faecalis isolates could also be expected to influence biofilm formation.

Stressful environmental conditions associated with root canal treatment may increase the expression of

cell-surface proteins and hydrophobicity, thereby improving attachment of bacteria to surfaces (Zilm

& Rogers 2007). The findings of the present study therefore have potential implications for endodontic

treatment. Clinically, the use of Ca(OH)2 during root canal treatment produces a concentration gradient

of hydroxyl ions originating from the root canal and decreasing along the apical dentinal tubules.

Those bacteria sheltering deep within the dentinal tubules may therefore respond to the changing

stressful environment by up-regulating virulence genes promoting biofilm growth (Love 2001, Bryce

et al. 2009). The concentration of other endodontic medicaments and irrigants also declines as they

diffuse through the complex anatomy of the root canal system (Nair et al. 2005) and components of

dentine can also partially inactivate chemicals such as NaOCl (Haapasalo et al. 2003). For some of the

isolates tested, sub-lethal concentrations of antimicrobial would be effective in inhibiting biofilm

growth. However, given the 3240 phenotype, the presence of NaOCl and tetracycline at inadequate

concentrations could improve the pathogens overall survival. As such, the findings support previous

studies showing that antimicrobials must be used at sufficiently high concentrations to be effective

against E. faecalis (Gomes et al. 2001).

A possible explanation for the differences in biofilm formation shown by the E. faecalis isolates could

be their ability to produce extracellular slime. The present finding that 80% of the isolates were either

strong or weak slime producers, supports that of Kouidhi et al. (2011) who found that 71% of E.

faecalis isolates produced slime. Previous studies have linked slime production with bacterial

adherence to surfaces and resistance to antimicrobials (Christensen et al. 1982, Farber et al. 1990).

When comparing slime production with antibiotic resistance, the two traits did not appear to be

correlated. Whilst isolates 3240 and the JKD 15036 were strong and weak slime producers,

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 respectively, they were found to both be sensitive to tetracycline and clindamycin. Isolates that were
Accepted Article weak slime producers (3326, 3325 and V583) also varied in their sensitivities to the antibiotics. In

contrast, the ability of the isolates to produce slime did appear to correlate with their capacity to form

a biofilm in the presence of the irrigants and medicaments tested. An optimized method suggested by

Arciola et al. (2002), using a colorimetric scale with six categories for greater differentiation between

isolates, could be used for any further studies to assess slime production amongst E. faecalis isolates.

CONCLUSION

Clonal variation among isolates of E. faecalis affected biofilm formation in the presence of sub-MIC

concentrations of endodontic medicaments and irrigants. Studies on the resistance of E. faecalis to

endodontic antimicrobials by only a single isolate or several isolates may not give a complete

understanding of bacterial resistance during root canal treatment. Further studies aim to identify what

causes the differences and consequently how best to target these factors. Based on the results from the

present study, investigations are on-going on whether clonal variation amongst E. faecalis may

provide an explanation as to why root canals treated under optimal conditions can have varying

results.

ACKNOWLEDGEMENTS

We would like to thank Michelle Lorimer from the University of Adelaide Data Management and

Analysis Centre for additional assistance with the statistical analysis while N. Briggs was on maternity

leave.

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Figure legends

Figure 1. Biofilm growth of E. faecalis isolates in 0.059% NaOCl expressed as estimated mean

optical density (OD595nm) and 95% CI. * Statistical significance, p-values were adjusted for multiple

comparisons, 3240 (p = 0.018), all other isolates p < 0.0001. Control was un-inoculated.

Figure 2. Biofilm growth of E. faecalis isolates in sub-inhibitory concentrations of CHx expressed as

estimated mean optical density (OD595nm) and 95% CI. * Statistical significance, p-values were

adjusted for multiple comparisons, p < 0.001 for all isolates. Control was un-inoculated.

Figure 3. Biofilm growth of E. faecalis isolates in 0.16% Ca(OH)2 expressed as estimated mean

optical density (OD595nm) and 95% CI. * Statistical significance, p-values were adjusted for multiple

comparisons, V583, p < 0.001; JKD 15036, p = 0.020; 3326, p = 0.035. Control was un- inoculated.

Figure 4. Biofilm growth of E. faecalis isolates in sub-inhibitory concentrations of clindamycin

expressed as estimated mean optical density (OD595) and 95% CI. * Statistical significance, p-values

were adjusted for multiple comparisons, JKD 15036, 3326, p < 0.0001. Isolate V583 was omitted as it

was resistant to clindamycin. Control was un-inoculated.

Figure 5. Biofilm growth of E. faecalis isolates in sub-inhibitory concentrations of tetracycline

expressed as estimated mean optical density (OD595) and 95% CI. * Statistical significance, p-values

were adjusted for multiple comparisons, V583 and 3240, p < 0.0001. Isolates 3326, 3325 were omitted

as they were resistant to tetracycline. Control was un-inoculated.

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 Figure 6. Growth of E. faecalis on Brain Heart Infusion agar containing Congo red. Isolate 3240 was
Accepted Article positive for slime production forming black pigmented colonies (A), weak slime producers (3325,

3326 and V583) displayed partially black pigmented colonies (B), isolate JKD 15036 was not able to

produce slime and produced no pigment (C).

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Accepted Article

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Accepted Article

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Accepted Article

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