UNIVERSITE DE GENEVE FACULTE DE MEDECINE Section de Médecine dentaire Département de prévention et thérapeutique Division de Médecine dentaire préventive
Thèse préparée sous la direction du Professeur Pierre BAEHNI
Effets d’agents antimicrobiens sur un modèle de
biofilm dentaire in vitro
THESE
présentée à la Faculté de Médecine de l’Université de Genève pour obtenir le grade de Docteur en médecine dentaire
par Hiroyuki Takinami de Fukui (Japon) Thèse N° 654 Genève 2007
UNIVERSITE DE GENEVE FACULTE DE MEDECINE Section de Médecine dentaire Département de prévention et thérapeutique Division de Médecine dentaire préventive
Thèse préparée sous la direction du Professeur Pierre BAEHNI
Effets d’agents antimicrobiens sur un modèle de
biofilm dentaire in vitro
THESE
présentée à la Faculté de Médecine de l’Université de Genève pour obtenir le grade de Docteur en médecine dentaire
par Hiroyuki Takinami de Fukui (Japon) Thèse N° 654 Genève 2007
TABLE DES MATIERES
1. RESUME FRANÇAIS p. 3
2. ABSTRACT p. 7
3. INTRODUCTION p. 8
4. MATERIALS & METHODS p. 12
5. RESULTS p. 15
6. DISCUSSION p. 18
7. ACKNOWLEDGEMENTS p. 27
8. REFERENCES p. 28
RÉSUMÉ FRANÇAIS
La flore orale est constituée d’une grande diversité de microorganismes. Elle comprend des
virus, des bactéries, des champignons, des mycoplasmes et, parfois, des protozoaires. Les
bactéries, représentées par plus de 500 espèces, occupent, au sein de cet ensemble, la part la
plus importante. Les microorganismes qui colonisent les surfaces dentaires s’organisent en
biofilm, appelé plaque dentaire. Le biofilm dentaire se compose de différentes populations
bactériennes entourées d’une matrice de polysaccharides et de glycoprotéines.
La prévention des maladies associées à la présence de biofilms, telles que la carie dentaire ou
les maladies parodontales, passe par le contrôle de la plaque dentaire. Celui-ci est réalisé le
plus souvent par des moyens mécaniques, tel que le brossage, mais aussi par des moyens
chimiques. Les agents chimiques sont utilisés surtout sous forme de bains de bouche, en
complément des moyens mécaniques. Les agents antiplaques se définissent par leur capacité de
perturber la plaque dentaire et ainsi prévenir le développement de gingivite ou de carie dentaire.
Tous les agents antimicrobiens n’ont pas nécessairement des propriétés antiplaques. Les agents
antiplaques actifs peuvent agir sur la formation du biofilm en interférant avec les mécanismes
vitalité bactérienne ou en perturbant le biofilm déjà organisé. De nombreux agents
antibactériens ont été testés dans des essais cliniques mais peu ont montré des propriétés
antiplaques.
Le digluconate de chlorhexidine (CHX) compte parmi les composés les plus éprouvés. Ses
propriétés antiplaques sont bien établies. La CHX est un bisbiguanide cationique qui interagit
avec les charges négatives de la pellicule acquise et inhibe l’adhésion bactérienne à la surface
amélaire. La CHX est également connue pour ses caractéristiques bactériostatiques vis-à-vis de
la majorité des bactéries orales. À haute concentration, elle devient bactéricide et agit tel un
détergent en endommageant les membranes bactériennes.
Le triclosan (TRI) est un composé phénolé anionique. Seul, il présente de faibles propriétés
inhibitrices sur la plaque dentaire, si bien qu’il est souvent associé à d’autres agents
antibactériens ou à des agents qui augmentent sa rémanence, comme le pyrophosphate, le
citrate de zinc, les huiles de silicone ou des copolymères.
L’isopropyl-méthylphénol (IPMP) est également un composé phénolé anionique, isomère du
thymol. On le retrouve dans divers bains de bouche en raison de son activité antibactérienne et
Le chlorure de cétylpyridinium (CPC) constitue un groupe d’agents tensioactifs cationiques.
Tandis que le CPC s’est révélé particulièrement efficace in vitro en tant qu’agent antiseptique,
son efficacité sur la plaque dentaire et sur la réduction de la gingivite reste secondaire.
L’objectif principal de cette étude consistait à évaluer in vitro l’effet de ces agents
antimicrobiens sur un modèle de biofilm. Un deuxième objectif était de comparer l’effet de ces
agents selon que le biofilm était immature ou mature. Dans le modèle utilisé, des disques
d’hydroxyapatite sont incubés en présence de 4 espèces bactériennes orales – Actinomyces
naeslundii, Fusobacterium nucleatum, Streptococcus oralis et Veillonella dispar – dans un
milieu anaérobie pendant 12 heures (biofilm immature) ou 73 heures (biofilm mature).
L’exposition aux agents antimicrobiens a été réalisée à 4, 8 et 12 heures pour le biofilm
immature et à 65, 69 et 73 heures pour le biofilm mature. Les biofilms ont été mis en présence
de 0,2 % de CHX, 0,2 % de TRI, 0,05 % de CPC et 0,1 % d’IPMP. Des groupes contrôles ont
été réalisés en exposant les biofilms à des solutions de 0,9 % de NaCl et de 10 % de C2H5OH.
Après exposition, les biofilms ont été recueillis et le nombre total de bactéries a été évalué par
culture sur gélose (nombre d’UFC). Certains prélèvements ont été observés au microscope
morphométrique de la structure du biofilm a été réalisée avec un logiciel de reconstruction
tridimensionnelle.
La CHX et l’IPMP provoquèrent une diminution significative des UFC par rapport aux
contrôles aussi bien avec le biofilm immature que mature, tandis que le TRI n’eut un effet que
sur le biofilm mature. Le CPC eut peu d’effet sur les deux types de biofilm. L’observation au
microscope confocal a montré que l’épaisseur du biofilm fut significativement plus élevée pour
les biofilms matures par rapport aux biofilms immatures. En présence de CHX, l’épaisseur du
biofilm immature était significativement plus faible par rapport aux autres groupes. L’analyse
3D mit en évidence que l’effet bactéricide était plus marqué à la surface du biofilm et que des
ABSTRACT
The aim of the study was to assess the effect of antimicrobial agents on an in vitro dental
biofilm model using culture and confocal laser scanning microscopy (CLSM). Hydroxyapatite
discs were incubated with four oral bacterial species (Actinomyces naelundii, Veillonella dispar,
Streptococcus oralis, Fusobacterium nucleatum) in anaerobic conditions for 12 (early biofilm)
or 73 hrs (mature biofilm). Biofilms were exposed to 0.2% chlorhexidine (CHX), 0.2%
triclosan (TRI), 0.05% cetylpyridinium chloride (CPC), and 0.1% isopropyl methylphenol
(IPMP), at various time intervals. Bacteria were harvested, and the total number of CFUs was
determined by culture. Specimens were observed by CLSM following live/dead fluorescence
staining and morphometry of the biofilm structure was analyzed using extensive
three-dimensionally reconstructing software. For early biofilm, CHX and IPMP resulted in a
significant decrease in CFUs compared to controls. The thickness of the biofilm in the CHX
group was significantly different than controls. For mature biofilm, a significant reduction was
also observed with CHX, TRI, and IPMP compared to controls. CPC had little effect on both
early and mature biofilms. 3D analysis revealed that killing occurred mostly in the superficial
INTRODUCTION
The resident oral microflora consists of a great diversity of microorganisms, including viruses,
bacteria, fungi, mycoplasma and, in some instances, protozoa (Baehni and Takeuchi, 2003).
Bacteria represent the predominant component of the oral microflora and may include up to
500 different bacterial species (Paster et al., 2001).
Dental plaque that grows on the tooth surface is organized as a biofilm. It is composed of
different bacterial populations embedded in a matrix of exopolysaccharides and glycoproteins
(Costerton et al., 1987; Listgarten, 1999). The aim of controlling the development of dental
plaque is to prevent biofilm-associated diseases such as dental caries and periodontal diseases
(Keyes and Jordan, 1964; Löe et al., 1965; Lindhe et al., 1973). Control of dental plaque may
be achieved by mechanical measures as well as by chemical agents. Chemical agents should be
used as adjuncts to mechanical plaque control. Anti-plaque agents are defined as chemicals that
have an effect on plaque sufficient to prevent gingivitis and/or caries (Lang and Newman,
1997). Not all antimicrobial agents have anti-plaque properties. Active anti-plaque agents
This can be achieved by interfering with bacterial adhesion and co-aggregation mechanisms,
by affecting bacterial vitality or by disrupting existing biofilms (Baehni and Takeuchi, 2003). A
variety of different antimicrobial compounds have been tested in clinical trials and are used in
practice (Mandel, 1988).
Chlorhexidine digluconate (CHX) is among the most studied compounds, and its anti-plaque
properties are well established (Fardal and Turnbull, 1986; Addy and Moran, 1997; Baehni and
Takeuchi, 2003). CHX is a cationic bisbiguanide that adsorbs to negatively charged sites of the
acquired pellicle present on the enamel surface and inhibits bacterial adhesion (Rölla and
Melsen, 1975; Pratten et al., 1998). CHX is also known to be bacteriostatic against most oral
bacteria (Stanley et al., 1989). In a supragingival biofilm model, CHX was shown to inhibit
bacterial growth and biofilm formation (Guggenheim et al., 2001; Shapiro et al., 2002). At
high concentrations, the agent is bactericidal and acts as a detergent by damaging the bacterial
cell membrane (Meurman, 1988). Triclosan (TRI) is a non-ionic phenolic compound. Because
TRI alone has only moderate plaque-inhibitory properties (Saxton, 1986; Jenkins et al., 1989),
it is often combined with other antibacterial agents or with agents that enhance its activity, e.g.,
Isopropyl methylphenol (IPMP) is also a non-ionic phenolic compound. IPMP, a constitutional
isomer of thymol, is found in several commercially available mouthrinses because it has
antibacterial activity. In addition, IPMP has high penetrability properties (Morishima et al.,
2004). Cetylpyridinium chloride (CPC) is a group of cationic surface-active agents. Although
CPC is very effective as an antiseptic agent in vitro, its effectiveness in plaque and gingivitis
reduction has been marginal (Addy and Moran, 1989; Moran and Addy, 1991).
Different techniques, including light microscopy, scanning, and transmission electron
microscopy, have been used in studies of microbial biofilms (Listgarten, 1976; Brecx et al.,
1981; Rimondini et al., 1997; Hannig, 1999). Besides these established techniques, confocal
laser scanning microscopy (CLSM) has been used more recently. It offers the possibility of
detailed visualization of “thick” microbiological samples in cases where the application of
conventional phase contrast or fluorescence microscopy is limited. CLSM eliminates
out-of-focus haze and allows horizontal and vertical optical sectioning. In dentistry, CLSM has
often been used for the investigation of hard mineral surfaces (Van der Veen and Ten Bosch,
1995; Øgaad et al., 1996). It has now become a powerful tool in microbiology for assessing
2001).
The primary objective of the present study was to assess the effect of selected antimicrobial
agents on biofilm in vitro. Another objective was to compare their effects on early and mature
biofilms. Specimens were evaluated by culture methods as well as by CLSM using a 3D
MATERIALS & METHODS
Preparation of biofilms
Biofilms were established according to the Zürich biofilm model (for detailed protocol, see
Guggenheim et al., 2004). The model is based on a batch culture approach and not on a
continuous flow culture system. Actinomyces naeslundii OMZ 745, Veillonella dispar OMZ
493, Streptococcus oralis OMZ 607, and Fusobacterium nucleatum ATCC 25586 were used as
inocula for biofilm formation (Guggenheim et al., 2001; Shapiro et al., 2002; Thurnheer et al.,
2003). Strains were maintained under anaerobic conditions on Columbia agar plates with sheep
blood (CBA; Oxoid GmbH, Wesel, Switzerland). Sintered hydroxyapatite discs (HA)
(Clarkson Chromatography Products Inc., South Williamsport, PA, USA) placed in 24-well
polystyrene cell culture plates (Becton Dickinson and Company, Franklin Lakes, NJ, USA)
were first coated with pooled saliva for 4 hrs (Guggenheim et al., 2001; Shapiro et al., 2002;
Thurnheer et al., 2003) and covered with 1.6 ml of 50% saliva-50% modified universal fluid
medium (mFUM) (67 mmol of Sørensen buffer/liter (final pH, 7.2) and 0.3% (wt/vol) glucose)
(Gmür and Guggenheim, 1983). Wells were then inoculated with mixed bacterial suspensions
species, 1.0 ± 0.05 [range of acceptable values]) and incubated anaerobically at 37°C for 12 hrs for early biofilms and 73 hrs for mature biofilms. For mature biofilms, discs were transferred to
new plates after 12 hrs of incubation and immersed in 1.8 ml of 50% saliva- 50% mFUM
containing 0.15% (wt/vol) glucose and 0.15% (wt/vol) sucrose and further incubated for 56
hrs.
Antimicrobial agents
Chlorhexidine digluconate (CHX), cetylpyridinium chloride (CPC), isopropyl methylphenol
(IPMP) and triclosan (TRI) were purchased from Fluka (Buchs, Switzerland). All agents were
diluted with 10% ethanol (Et-OH).
Exposure of biofilms to antimicrobials
Early and mature biofilms were immersed in 1 ml of antibacterial solution for 1 min and rinsed
gently by dipping the discs 3 times in 2 ml of physiological saline. Exposure to antimicrobial
agents was performed at 4, 8, and 12 hrs for early biofilm and at 65, 69, and 73 hrs for mature
biofilm. 0.9% NaCl and 10% Et-OH were used as negative controls. After the last exposure,
biofilms were harvested by vigorous vortexing in physiological saline (1 ml). Bacterial
agar plates (FAA) using a Spiral plater system (Spiral Systems Inc., Cincinnati, OH, USA).
Plates were incubated anaerobically at 37°C for 72 hrs, and the number of colony-forming units (CFUs) was determined with the aid of a stereomicroscope.
Confocal Laser Scanning Microscopy
For morphological evaluation, specimens were stained with 1 ml saline solution containing 5
µl of fluorescein diacetate (FDA) and 5 µl ethidium bromide (EB). Specimens were then transferred to a confocal laser scanning microscope (LSM 510 META, Zeiss, Germany),
placed upwards and examined with an argon (514/488 nm) and HeNe (543 nm) laser using a
40x objective lens with water immersion. The thickness of each section was 1.0 µm. Biofilm thickness was measured on cross-sections from five randomly selected positions using the
LSM Image Browser (Zeiss, Germany). 3D digital images were processed using Imaris 4.2
software (Bitplane, Zurich, Switzerland).
Statistical Analysis
Total CFUs were log transformed to fit a normal distribution. Biofilm thickness was presented
as mean values (µm) ± SE. Statistical analysis was performed with the Kruskal-Wallis test (StatView J5.0; Abacus Concepts, Inc., Berkeley, CA, USA) for comparison between groups.
RESULTS
Results from cultures
In early biofilms, CFU median values for controls were between 3.3 x 109 and 6.0 x 109
(Fig.1A). Exposure to CHX and IPMP resulted in a significant reduction in the number of
CFUs compared to controls (p < 0.05) : CFU median values decreased to 7.8 x 108 (76.4%) and
7.4 x 108 (77.6%) respectively. Values observed with TRI (1.4 x 109) and CPC (3.2 x109) were
not significantly different from controls. In early biofilms, V. dispar and S. oralis were the
predominant species, whereas F. nucleatum was not detected (Fig.2A). No major differences
in the bacterial composition were observed between the different groups except with CHX,
where S. oralis was the only detected species. In mature biofilms, CFU median values for
controls were between 8.9 x 1013 and 9.6 x 1013 (Fig.2B). A significant reduction in the number
of CFUs was observed with CHX (2.1 x 1012), TRI (2.4 x 1012) and IPMP (2.9 x 1012)
compared to controls (p<0.05), corresponding approximately to a 97 % reduction. CFU values
were also found to be lower with CPC (4.7 x 1012), although the results were not statistically
different from the controls. S. oralis was the most frequently detected species in mature
different groups.
Morphological observations
Following staining with fluorescein diacetate and ethidium bromide, viable bacteria appear
green, whereas dead bacteria are red. Fig.3 shows IMARIS imaging of the biofilm models
constructed from confocal images. In control specimens, in both early and mature biofilms, one
can see that the majority of bacteria were viable: most cells appeared green. In early biofilm,
substantial killing occurred in specimens exposed to CHX and IPMP, as suggested by the high
proportions of red cells. Significant proportions of dead cells were also observed in TRI-treated
biofilms. Dead bacteria were seen mostly on the surfaces of biofilms, covering layers of green
cells on the bottom. In specimens exposed to CPC, most bacteria were viable with only few
aggregates of red cells present. For mature biofilms, substantial bacterial killing was evident
with all four antimicrobials tested. The effect was particularly evident following exposure to
CHX and TRI. In specimens treated with IPMP and CPC, viable bacteria were clearly visible at
the bottom of the biofilms
(Fig. 3B).
thickness was significantly higher in mature biofilms than in early biofilms. In control
specimens, the thickness was between 15.0±3.7 and 13.6±2.3 µm and 36.8±2.1 and 37.6±1.5 µm, respectively, for early and mature biofilms. There was no significant difference in thickness between specimens exposed to antimicrobial compounds and controls, with the
DISCUSSION
The development of in vitro biofilm models represents a major advance because they allow a
better understanding of the nature of dental plaque and its physiological aspects as well as
interactions among microbial species within biofilms. Organisms that compose biofilms are not
merely passive neighbors but are involved in a wide range of physical, metabolic and
molecular interactions (Marsh, 2005). Bacteria adhere by adhesion–receptor interactions either
to the acquired pellicle or to already attached cells (co-adhesion). Bacteria interact
synergistically to metabolize complex host molecules, and food webs can develop, enabling
the efficient cycling of nutrients. Within biofilms, sophisticated systems of cell–cell
communication are used by some bacteria to co-ordinate gene expression. Gram-positive
bacteria generally communicate via small diffusible peptides (Sturme et al., 2002), whereas
many Gram-negative bacteria secrete acyl homoserine lactones (Whitehead et al., 2001), the
structure of which varies depending on the species of bacteria that produce them.
In vitro biofilm models previously described have used different culture systems, such flow
cells (Christersson et al., 1987; Larsen and Fiehn, 1996; Sjollema et al., 1989) or chemostats,
al., 1996; Herles et al., 1994; Kinniment et al., 1996). The use of flow cells and chemostat
devices are not suitable for studying the effect of antimicrobial agents on biofilms because
clearance of pulsed substances is a function of flow rate and volume. Chemostats operating
with low rates and relatively large volumes can have quite long mean residence times,
rendering them impractical for studies of selected compounds with short-term exposures, as is
common in oral hygiene procedures. Moreover, systems with working volumes of more than a
few milliliters preclude the use of liquids constituted from natural substrates, such as saliva.
Therefore, the model used in the present study was based on a batch culture approach. Bacterial
species included two Gram positive species (S. oralis and A. naeslundii) and two Gram
negative species (V. dispar and F. nucleatum). These species are considered early colonizers in
the development of dental biofilm and are frequently detected in supragingival plaque. This
model was selected because the target of antimicrobials, when used as mouthrinses, is mainly
supragingival dental plaque. It was interesting to note that F. nucleatum was not detected in
early biofilm. This suggests that F. nucleatum is a later colonizer than the other species used in
It has been shown that bacteria within biofilm possess different characteristics and properties
than planktonic cells. For example, cells within biofilms have been shown to be less
susceptible to antimicrobial agents (Gilbert et al., 1997). Indeed, the minimum inhibitory
concentration (MIC) of CHX was shown to be 300 times greater when S. sobrinus was grown
as a biofilm than when planktonic cells were grown (Shani et al., 2000). Similarly, S. sanguis,
re-named S. sanguinis, in biofilms resisted 10–50 times the MIC of CHX for up to 24 hrs
(Larsen and Fiehn, 1996). Another study showed that repeated exposures of mixed culture oral
biofilms to CHX were effective only at concentrations 10 times the MIC (Kinniment et al.,
1996). This may be related to physical properties of the biofilm or to protection of bacterial
clusters by neighboring cells (Marsh and Bowden, 2000). Studies using confocal laser
scanning microscopy showed that, in established biofilms exposed to antimicrobials, bacterial
killing occurred mostly in superficial layers, suggesting either quenching of the agent at the
biofilm surface or a lack of penetration (Zaura-Arite et al., 2001). In addition, environmental
heterogeneity generated within biofilms encourages genotypic and phenotypic diversity, which
enhances bacterial ability to persist in the face of assault from innate and adaptive immune
Costerton, 2004).
Early electron microscopic studies reported that dental plaque was composed of dense bacterial
masses embedded in a matrix (Listgarten, 1976). More recent studies have shown that
supragingival plaque is a more open structure with channels traversing from the surface of the
biofilm to the enamel surface (Wood et al., 2000; Auschill et al., 2001; Zaura-Arite et al.,
2001). Our observations are in agreement with these reports. Pictures obtained by CLSM
showed non-stained structures within the biofilm architecture. It is assumed that nutrients can
diffuse through the pores or the channels within the biofilm. Its structure may also have
important implications for the penetration and distribution of molecules, such as antibacterial
agents, within plaque. A study on the diffusion of macromolecules (3,000 [3K-Dex] to
240,000) through an in vitro biofilm model (Thurnheer et al., 2003) showed a linear
relationship between the molecular weight, ranging from 10,000 to 240,000, and the depth of
penetration. These observations suggest that diffusion of molecules is affected by the biofilm
matrix and the convoluted paths. A dramatic increase in diffusivity, however, occurred as the
probe molecular weight decreased from 10,000 (10K-Dex) to 3,000. This was attributed to
narrower than 4.6 nm (estimated diameter of 10K-Dex). It should be noted that the molecular
mass for the CHX (897.8 Da), TRI (289.5 Da), CPC (385.8 Da), and IPMP (150.2 Da) used in
the present study are much smaller than 3,000, suggesting a high diffusion rate. This is in
agreement with the results reported elsewhere (Hope and Wilson, 2004).
Observations following live/dead staining suggest that bacterial vitality may vary throughout
the biofilm (Netuschil et al., 1998), with higher numbers of viable bacteria in the central part
of the plaque or in the lining of the channels (Auschill et al., 2001). In preliminary experiments,
we used BacLight (Molecular Probes, Eugene, OR, USA) for determining bacterial viability.
BacLight is a popular fluorescence-based two-component stain for detecting live/dead bacteria.
However, artifacts may occur if the relative intensities of the stains or the concentration of
propidium iodide relative to nucleic acid are not properly accounted for (Stocks, 2004). Instead,
we used FDA-EB for vital staining. FDA is a fluorogenic substance capable of penetrating cell
membranes. It is cleaved enzymatically in the cytoplasm, leaving fluorescent fluorescein,
which cannot leave the living cell through its membrane. EB does not penetrate intact cell
membranes but stains the DNA and RNA of dead cells. This method has proved useful for
(red) bacteria (Auschill et al., 2001; Netuschil et al., 1989).
Pictures obtained by CLSM should be interpreted with caution because they show compressed
images of biofilm structures (Fig.3B). In the early biofilm experiments, one may assume that
the images show most of the bacteria present in the observed field because bacterial density
was low (Fig.3A, 3C). In some instances, images suggest the presence of a layer of living
bacteria on the bottom of the biofilm (Fig. 3B). However, analysis of the same field on a 3D
image revealed that this was an artifact and that only a few clusters of live cells were present
(Fig. 3A, 3C). On the other hand, in mature biofilms, when the bacterial density increased,
images revealed only part of the biomass up to a certain depth. In this respect, 3D image
analysis proved to be valuable tool because it provided additional information.
The aim of the present investigation was to evaluate the effect of different antimicrobials and to
compare their effects on early and mature biofilms. NaCl and Et-OH served as controls
because 10% Et-OH was used to dissolve the antibacterial agents tested. Results showed that
Et-OH did not affect the biofilms when compared to saline. Other studies showed that Et-OH
concentrations higher than 40% can inhibit growth of plaque biofilms but that 10% Et-OH has
decrease in the number of CFUs. One should note that the magnitude of the effect was modest
with a 1 log reduction compared to controls. In contrast, observations by CLSM suggest that
large proportions of the bacterial biomass were composed of dead cells as indicated by vital
staining. The reason for the discrepancy between culture data and CLSM observations remains
unknown. CHX was the most effective agent among the four antimicrobial agents tested in
both early and mature biofilms. This was shown by culture methods as well as by CLSM. At
low concentrations, CHX causes damage to cell membranes allowing low-molecular-weight
molecules to escape from the bacteria (Jones, 1997). At high concentrations, CHX causes
precipitation and coagulation of the proteins in the cytoplasm of the bacteria. The MIC of CHX
against oral bacteria was reported to be between 8 and 500 µg/ml (0.0008–0.05% w/v) (Stanley et al., 1989). In clinical practice, CHX is usually prescribed at concentrations of
0.1–0.2%. There is ample clinical evidence that CHX, at 0.2% or less, is highly predictable in
reducing supragingival plaque accumulation and gingival inflammation (Addy and Moran,
1997). In the present study, CHX was used at a 0.2% concentration. The effect of CHX on
plaque development was supported by the fact that the thickness of the early biofilm was
shown previously by CLSM in another in vitro study (Auschill et al., 2005). One may
hypothesize that CHX molecules initially adsorb onto the salivary pellicle or the biofilm
surface and remain active so that bacterial adhesion is inhibited (Rölla and Melsen, 1975;
Pratten et al., 1998). CLSM following vital staining revealed that live bacterial clusters were
still present after exposure to CHX. It is probable that a 1 min exposure time was too short to
kill all bacteria, even in early biofilms, and that the remaining bacteria were able to develop
into a biofilm structure. S. oralis was the only detected species in early biofilm exposed to
CHX. The exact mechanism is unknown but it is possible that CHX may have interfered with
the co-adhesion of other bacterial species to S. oralis.
TRI is bacteriostatic at low concentrations and is bactericidal at high concentrations. IPMP
causes protein denaturation and cell membrane damage whereas CPC generally affects cell
membrane (Sreenivasan and Gaffar, 2002). In our experiments, TRI was less effective than
CHX for early biofilms but was comparable to it for mature biofilms. In contrast, the effect of
IPMP was similar to that of CHX in early biofilms but was lower than CHX in mature biofilms.
This may be related not only to the antibacterial activity of the agents but also to the diffusion
values for P. gingivalis, A. viscosus, F. nucleatum, and V. parvula than CHX, TRI, and CPC but
it has a greater potential of penetrability (Morishima et al., 2004). One may hypothesize that a
1 min exposure time may have been too short for IPMP to diffuse and alter bacterial vitality in
mature biofilm compared to agents with lower MIC values.
The substantivity of the tested agents varies markedly (Cummins, 1992). Approximately 32%
of the CHX dose from a range of vehicles (mouthwashes, gels, etc.) is retained in the mouth.
The uptake and release of CHX is highly pH dependent (e.g., uptake is reduced at low pH) and
is inhibited by calcium ions. Similar levels of TRI are retained immediately after toothbrushing
(25%), but subsequent rates of clearance vary significantly. The half-life for clearance of
bound TRI is about 20 min. However, TRI could be detected in plaque for at least 8 hrs after
toothbrushing (Gilbert and Williams, 1987). In contrast, quaternary ammonium compounds
such as CPC have only low levels of substantivity, which reduces their effectiveness under the
normal conditions of use in the mouth.
In conclusion, the present study investigated the effect of four antimicrobial agents on early
and mature biofilms using culture methods and CLSM. The results confirmed a discrepancy
bacteria. CLSM allowed confirmation that penetration of antimicrobial agents into the bottom
of biofilms was difficult. 3D images proved to be a valuable tool for studying the effect of
ACKNOWLEDGEMENTS
We would like to thank J. Ritz, C. Bauer (Bioimaging Platform, Frontiers in Genetics NCCR,
Geneva) S. Arnaudeau and O. Brun (Bioimaging core facility, University of Geneva) for help
with CLSM and T. Thurnheer and B. Guggenheim (Oral Microbiology and General
Immunology, University of Zürich) for valuable technical advice. We also wish to thank A.
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Table1 Biofilm thickness after 12 and 72.5 hours plaque formation
Mean ± SD values (µm)
NaCl Et-OH CHX TRI CPC IPMP Early Biofilm 15.0±3.7 13.6±2.3 8.2±1.8* 9.0±1.6 12.0±1.4 10.0±1.2 Mature Biofilm 36.8±2.1 37.6±1.5 34.2±1.5 33.8±1.8 35.6±0.9 35.0±2.6
Table1
LEGENDS
Fig.1
Boxplots depicting total viable cell count in early and mature biofilms. Number of
observations: n = 12. Each box indicates the lower and upper quartiles; the central line is the
median; whiskers indicate the maximum and minimum values. Significant differences between
treatments:
*** p < 0.001, * p < 0.05
Fig.2
(A): Number of CFU for each bacterial species. (B): Percentage of each bacterial species.
Fig.3
Three-dimensional reconstruction of biofilm by IMARIS software.
Early biofilm 1.0E+07 1.0E+08 1.0E+09 1.0E+10 1.0E+11 1.0E+12 1.0E+13 1.0E+14 1.0E+15
NaCl Et-OH CHX TRI CPC IPMP
CFU
*
*
A
Mature biofilm 1.0E+07 1.0E+08 1.0E+09 1.0E+10 1.0E+11 1.0E+12 1.0E+13 1.0E+14 1.0E+15NaCl Et-OH CHX TRI CPC IPMP
CFU
***
***
*
B
Fig.1
Early biofilm 0% 20% 40% 60% 80% 100%
NaCl Et-OH CHX TRI CPC IPMP
% of bacteri a l speci es S.o V.d A.n F.n
A
B
Fig.2
Mature biofilm 0% 20% 40% 60% 80% 100%NaCl Et-OH CHX TRI CPC IPMP
% of i ndi vi dual speci es S.o V.d A.n F.n Mature biofilm 1.0E+10 1.0E+11 1.0E+12 1.0E+13 1.0E+14 1.0E+15
NaCl Et-OH CHX TRI CPC IPMP
CFU S.o V.d A.n F.n Early biofilm 1.0E+06 1.0E+07 1.0E+08 1.0E+09 1.0E+10 1.0E+11
NaCl Et-OH CHX TRI CPC IPMP
CFU
