In vitro inhibition of Streptococcus mutans biofilm formation on hydroxyapatite
by sub-inhibitory concentrations of anthraquinones
5
Tom Coenye1*, Kris Honraet1,2, Petra Rigole1,2, Pol Nadal Jimenez1 & Hans J. Nelis1
Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent1, and Oystershell NV,
10
Drongen2, Belgium
*
Corresponding author. Mailing address : Laboratorium voor Farmaceutische Microbiologie,
Universiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium. Phone : +32 9 2648093. Fax : + 32 9
15
2648195. E-mail : Tom.Coenye@UGent.be
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Running title : Inhibition of S. mutans biofilm formation by anthraquinones
1
ABSTRACT
We report that certain anthraquinones (AQ) reduce Streptococcus mutans biofilm formation
on hydroxyapatite in concentrations below the minimal inhibitory concentration. Although
5
AQs are known to generate reactive oxygen species, the latter do not underly the observed
effect. Our results suggest that AQs inhibit S. mutans biofilm formation by causing
membrane perturbation.
10
2
Dental plaque displays several properties typical of biofilms, including reduced
susceptibility to antimicrobial agents (9, 13). Streptococcus mutans is considered to be the primary
cariogen within dental plaque (12) and prevention or reduction of biofilm formation by S. mutans
could thus contribute to the prevention of caries. In the present study we evaluated the ability of
5
anthraquinones (AQs) to inhibit S. mutans biofilm formation in concentrations below the minimum
inhibitory concentration (MIC), and their mechanism of action.
S. mutans LMG 14558T, Micrococcus luteus NRRL B-2618 and Vibrio harveyi strains were
routinely grown in Brain Heart Infusion (BHI) broth (BD, Franklin Lakes, NJ) at 37°C, on TSA
(BD) at 30°C or on Difco Marine Agar (BD) at 37°C, respectively. The AQs tested are listed in
10
Table 1. To determine the MIC of each compound, a microdilution assay in 96 well microtiter
plates (TPP, Trazadingen, Switzerland) was used (8). S. mutans LMG 14558T biofilms were grown
on hydroxyapatite disks in Modified Robbins Devices (MRDs) and the biofilm biomass on each
disk was estimated using fluorescent staining with SYTO9 (Invitrogen, Carlsbad, CA) (8).
Inhibition of glucosyltransferase (GTF) by AQs was assessed by an enzymatic assay (16).
15
Induction of reactive oxygen species (ROS) by AQs was measured in two separate assays. In a first
assay, MICs were determined in the presence or absence of 1.5 mM glutathione (GSH) (Sigma),
0.025% (w/v) cysteine (Sigma) and 10 mM mannitol (Sigma), using the modified microdilution
assay described above. In a second assay we used the fluorescent probe 2’,7’-dichlorofluorescein
diacetate (DCF-DA) to quantitate the amount of ROS produced (5, 6). The inhibition of mutacin
20
production can be used as an indirect assay to detect competence-stimulating peptide (CSP)-based
quorum sensing (QS) (21). Mutacin production was determined using M. luteus NRRL B-2618 as
an indicator strain. Inhibition of autoinducer-2 (AI-2) based QS was studied using V. harveyi
biosensor strains, as described previously (13). Lateral diffusion of fatty acids in the cell membrane
was measured by the intermolecular excimerization of the fluorescent probe pyrene (2). To study
25
the rotational diffusion of the fatty acid acyl chains in the interior of the membrane, the
fluorescence anisotropy was measured using 1,6-diphenyl 1,3,5-hexatriene (DPH) (2). Cellular fatty
acid analysis was performed as described previously (20).
The overall mean fluorescence response for S. mutans biofilms grown on hydroxyapatite in
BHI supplemented with 1% sucrose (BHIS) (positive controls) (n = 386 disks) after staining with
30
SYTO9 was 13.22±2.83 x 105 relative fluorescence units, which correlates with approximately 5 –
6 x 108 cells per disk is (8). The MIC values for various AQs are shown in Table 1. Most of the
AQs tested in the present study exhibited no activity against planktonic S. mutans LMG 14558T
cells, with MIC values of ≥250 µg/ml (Fig. 1). The biofilms grown in the presence of emodin,
hypericin, carminic acid, chrysophanic acid or quinizarin (5 µg/ml) revealed a significant (P ≤
35
0.01) lower fluorescence response than biofilms grown in BHIS without AQs (Table 1). For
3
emodin, the most active compound, there was a quasi-linear relationship between its concentration
and relative biofilm formation (Table 1). None of the AQs investigated showed significant
inhibition of GTF (data not shown). The addition of 1.5 mM GSH, 10 mM mannitol or 0.025%
cysteine did not result in an altered MIC value for emodin in M1 medium (17) (Fig. 2). Similarly,
5
supplementing BHIS medium containing 5 µg/ml emodin with 0.025% cysteine did not result in
increased biofilm formation, i.e. cysteine had no protective effect against the action of emodin. This
strongly suggested that ROS generation is not the mechanism by which emodin affects S. mutans
cells. This was confirmed by using the oxidative stress – specific fluorescent probe DCF-DA (data
not shown). No effect of AQs was observed on CSP- or AI-2 - based quorum sensing (data not
10
shown). Incubation of S. mutans LMG 14558T with emodin or hypericin resulted in a decreased
membrane lateral and rotational diffusion (Table 2, Fig. 2), indicating a reduced membrane fluidity.
Although the average difference in anisotropy between DPH-labelled emodin-treated cells (0.1528)
and untreated cells (0.1327) was low (15.13%) and not significant (P = 0.512), these differences
were observed consistently. In addition, small changes (approximately 10%) in fluorescence
15
anisotropy may reflect marked changes (of about 25%) in membrane microviscosity (2, 11).
Together with the significant decrease in membrane lateral diffusion, the observed differences in
fluorescence anisotropy strongly suggest an effect of emodin on the membrane microviscosity.
It has previously been reported that membrane fluidity has an influence on many cellular
processes, including permeability, cold adaptation and growth and survival at suboptimal
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temperatures (3, 4, 10, 19). Bacterial membrane fluidity is most-often modulated by altering the
fatty acid composition but there were no significant differences in membrane fatty acid composition
between S. mutans cells grown in the presence or absence of emodin (data not shown). It had been
reported that emodin becomes inserted inside the phospholipid bilayer, strongly affects Van der
Waals interactions between hydrocarbon chains of phospholipids and destabilizes membrane
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bilayers by promoting non-bilayer phases (1). Based on this we suggest that the anti-biofilm effect
of emodin is caused by insertion of the planar molecule in the cell membrane and/or binding of that
same molecule to membrane-embedded molecules, including proteins. To our knowledge, there are
at present no data on the effect of changes in bacterial membrane fluidity on biofilm formation,
although an effect on adhesion potential appears plausible. The observation that the formation of a
30
S. mutans biofilm can significantly be reduced by AQs in sub-inhibitory concentrations is unusual
and may lead to novel strategies to prevent dental plaque and caries.
We thank D. P. Labeda, M. Uyttendaele and T. Defoirdt for providing strains, L. Vanhee for excellent
technical assistance, I. Vandecandelaere for the fatty acid analysis, and E. Lorent, Y. Engelborghs and S. Desmedt for
35
assistance with and helpful discussions regarding fluorescence polarisation. This work was supported by an IWT KMO
Innovation Project and by Oystershell NV (Belgium).
4
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6
Fig. 1. Susceptibility of S. mutans LMG 14558T to increasing emodin concentrations in various
media. MICs were determined using a modified microdilution assay in 96 well microtiter plates, as
previously described (8), in the presence and absence of 1.5 mM glutathione (GSH) (Sigma),
0.025% (w/v) cysteine (Sigma) and 10 mM mannitol (Sigma). These compounds are ROS
5
scavengers, protect cells from oxidative damage (18, 22) and will increase the MIC of emodin if its
antibacterial effect is due to ROS production. M1 is the chemically defined medium described in
reference 17. BHIS is BHI supplemented with 1% sucrose.
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7
Fig. 2. Fluorescence emission spectra of DPH-labelled S. mutans LMG 14558T cells grown in BHI
in the presence or absence of 5 µg/ml emodin. DPH is allowed to insert into the membrane, the
sample is subsequently excited with polarised light and the extent of polarisation of the emitted
light (which depends on the rotational Brownian motion) is measured. The magnitude of the
5
rotational diffusion of DPH depends on the temperature and the microviscosity (fluidity) of the
surrounding membrane. Cell suspensions were incubated with 5 x 10-6M DPH for 1 hour at 37°C,
and subsequently steady state fluorescence anisotropy was measured with a Photon Technology
International spectrofluorimeter (excitation with vertically polarised light of 360 nm, emission at
430 nm).
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15
8
Table 1. MIC values, concentrations used in biofilm experiments and effect of AQs. For
comparison we also determined the inhibition of biofilm formation by the antibacterial agents
triclosan and chlorhexidine, compounds commonly included as antibacterial agents in toothpaste
5
and mouthwash.
Compound
Emodin
Concentration
Relative biofilm
(µg/ml)
in MRD¶ (µg/ml)
formation (%)*
>250
5
10.9±0.9
<0.001
2
52.1±7.2
<0.001
1
79.0±10.9
0.005
0.5
85.7±12.7
NS
0.1
100.8±13.9
NS
10
15
20
25
30
Significance+
MIC
Hypericin
250
5
47.8±16.7
<0.001
Carminic acid
>250
5
66.5±23.15
0.005
Chrysophanic acid
250
5
75.0±10.0
<0.001
Rhein
10
5
75.2±25.0
NS
Quinizarin
250
5
80.0±17.2
0.01
Sennidin A
>250
5
83.7±17.8
NS
Chrysazin
250
5
91.3±9.3
NS
Anthraflavic acid
250
5
98.2±12.5
NS
Aloe-emodin
>250
50
99.2±22.2
NS
Physcion
>250
5
119.9±17.7
NS
Chlorhexidine
2
0.1
103.5±8.0
NS
1200
34.5±21.3
<0.001
5
78.13±5.5
0.005
1333
10.9±10.8
<0.001
Triclosan
16
¶
MRD, Modified Robbins’ device
*
average ± standard deviation (% compared to BHIS control)
+
P value for difference between biofilm formation in BHIS+product and BHIS (one-tailed
independent samples t test), NS : not significant (P > 0.01)
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Table 2. Relative fluidity of membranes of S. mutans grwon under different conditions, as
measured using pyrene and DPH. Data are presented as relative fluidity (% compared to BHI)
(average±standard deviation) and anisotropy (average±standard deviation), for pyrene and DPH,
respectively. At a constant absolute temperature T, the relative fluidity of the membranes of cells
5
treated with AQs (F) compared to the fluidity of membranes of untreated cells (Fr) is calculated as
follows : F = (Ie/Im) / (Ie/Im)r x Fr, with Ie/Im being the pyrene excimer-to-monomer fluorescence
intensity at temperature T (14, 15). For convenience, Fr (the relative fluidity of untreated S. mutans
cells grown in BHI) was set to 1. Fluorescence anisotropy is defined as A = I║ – I┬ / I║ + 2I┬ where
I║ and I┬ are the fluorescence intensities parallel and perpendicular to the direction of the excitation
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beam, respectively (2). % Difference in anisotropy values is calculated by the formula (ABHIAemodin)/Aemodin x 100. The green tea extract was selected as positive control as it was previously
shown that green tea catechins affect membrane fluidity (20)
Pyrene
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DPH
Fluidity
P-value
Anisotropy
BHI
100±27.8
-
0.1327±0.0433
-
BHI + 5 µg/ml emodin
21.2±47.4
<0.01
0.1528±0.0107
15.13
BHI + 5 µg/ml hypericin
35.6±28.2
<0.05
ND
ND
BHI + 1 % green tea
23.4±39.2
<0.05
ND
ND
20
10
% Difference