j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 9An in vitro study of alginate oligomer therapies onoral biofilms

J.L. Roberts a, S. Khan a, C. Emanuel a, L.C. Powell a,b, M.F. Pritchard a,b,E. Onsyen c, R. Myrvold c, D.W. Thomas a, K.E. Hill a,*aWound Biology Group, Tissue Engineering and Reparative Dentistry, Cardiff University School of Dentistry, Heath

Park, Cardiff CF14 4XY, UKbMultidisciplinary Nanotechnology Centre, School of Engineering, Swansea University, Swansea SA2 8PP, UKcAlgiPharma AS, Industriveien 33, N-1337 Sandvika, Norway

a r t i c l e i n f o

Article history:

Received 3 May 2013

Received in revised form

12 July 2013

Accepted 18 July 2013

Keywords:

Antimicrobial

Streptococcus mutans

Porphyromonas gingivalis

Triclosan

OligoG

a b s t r a c t

Objectives: The in vitro effect of a novel, oligosaccharide nanomedicine OligoG against oral

pathogen-related biofilms, both alone and in the presence of the conventional anti-bacterial

agent triclosan, was evaluated.

Methods: The effect of OligoG triclosan was assessed against established Streptococcusmutans and Porphyromonas gingivalis biofilms by bacterial counts and image analysis using

LIVE/DEAD1 staining and atomic force microscopy (AFM). The effect of triclosan and OligoG

surface pre-treatments on bacterial attachment to titanium and polymethylmethacrylate

was also studied.

Results: OligoG potentiated the antimicrobial effect of triclosan, particularly when used in

combination at 0.3% against S. mutans grown in artificial saliva. OligoG was less effective

against established P. gingivalis biofilms. However, attachment of P. gingivalis, to titanium in

particular, was significantly reduced after surface pre-treatment with OligoG and triclosan

at 0.01% when compared to controls. Light microscopy and AFM showed that OligoG was

biocidal to P. gingivalis, but not S. mutans.

Conclusions: OligoG and triclosan when used in combination produced an enhanced anti-

microbial effect against two important oral pathogens and reduced bacterial attachment to

dental materials such as titanium, even at reduced triclosan concentrations. Whilst the use

of triclosan against oral bacteria has been widely documented, its synergistic use with

OligoG described here, has not previously been reported. The use of lower concentrations of

triclosan, if used in combination therapy with OligoG, could have environmental benefits.

Clinical importance: The potentiation of antimicrobial agents by naturally occurring oligo-

mers such as OligoG may represent a novel, safe adjunct to conventional oral hygiene and

periodontal therapy. The ability of OligoG to inhibit the growth and impair bacterial

adherence highlights its potential in the management of peri-implantitis.

# 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +44 02920 744252; fax: +44 02920 742442.E-mail address: hillke1@cardiff.ac.uk (K.E. Hill).

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

0300-5712/$ see front matter # 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jdent.2013.07.011

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 9 893otherwise stated. Triclosan was solubilised in 3% (v/v)

isopropanol (with isopropanol controls included in all

in PBS and plated onto FAA to determine bacterial counts

(CFU/mL).1. Introduction

The microflora of the oral cavity contains >800 individual

species.1,2 The majority of these resident oral bacteria exist

in dynamic biofilms that do not usually impinge detrimen-

tally upon the normal health of a host. However, a variety of

host factors, such as poor dental hygiene and smoking,

can alter the ecological balance leading to the development

of pathologies such as dental caries, acute infection

or chronic periodontal inflammation.3 In the pathogenesis

of biofilm-related dental disease, considerable attention

has been directed towards the study of Streptococcus

mutans in the initial formation of carious lesions4 and

Porphyromonas gingivalis, associated with periodontitis,5 with

both organisms also shown to be implicated in peri-

implantitis.6

Alginates are linear polymers of (14) linked b-D-mannuro-

nic acid (M) and/or its C-5 epimer a-L-guluronic acid (G). They

are derived from marine algae and bacteria such as Pseudomo-

nas aeruginosa.7 The structure of OligoG has previously been

published.8 We have recently demonstrated that specifically

engineered (9095% G) low molecular weight alginate oligo-

mers potentiate the effectiveness of antibiotics against multi-

drug resistant bacteria (including Pseudomonas and Acineto-

bacter spp.); exhibiting both inhibition and disruption of

bacterial biofilms,8 as well as altering their mechanical

properties.9 Importantly, for potential clinical utilisation,

these agents are safe for human administration, biodegrad-

able and derived from a sustainable source. Although we have

shown that OligoG affects bacterial motility, other factors

contributing to its antimicrobial activity are still being

elucidated.

A common antimicrobial component within dental

products is triclosan (5-chloro-2-(2,4-dichlorophenoxy)phe-

nol) a broad-spectrum synthetic bisphenol.10 Triclosan is

effective against plaque bacteria; slowing the progression of

both caries and chronic periodontal disease.11 Whilst

triclosan is an effective antimicrobial against a range

of bacteria, environmental concerns exist regarding its

use at bacteriostatic (sub-MIC) concentrations, and its

relatively slow degradation which may lead to the develop-

ment of resistance.12 Furthermore, consumers are increas-

ingly eager to embrace the use of more natural

antimicrobials.13

We sought to determine whether the oligosaccharide

nanomedicine OligoG could potentiate the activity of triclosan

against two clinically relevant components of the dental

biofilm in health and disease; S. mutans and P. gingivalis.

2. Materials and methods

2.1. Chemicals and reagents

Bacteriological media was obtained from Oxoid (Basingstoke,

UK). OligoG (Algipharma AS, Sandvika, Norway) was dis-

solved in sterile phosphate buffered saline (PBS) unlessexperiments). Chlorhexidine 0.2% (v/v) was obtained from

GlaxoSmithKline, UK. Other chemicals were from Sigma

(Dorset, UK).

2.2. Bacteria

Bacterial strains employed were S. mutans DSM 20523 (ATCC

25175) and P. gingivalis NCTC 11834 (ATCC 33277).

2.3. Exposure of oral biofilms to OligoG and triclosan

Fastidious anaerobe broth (FAB) proved to be the most optimal

media for the growth of the highly fastidious P. gingivalis.

Although perhaps not optimal for the growth of S. mutans, it

was used in the biofilm assays with OligoG and triclosan to

enable direct comparison between both strains. S. mutans and

P. gingivalis were grown anaerobically in FAB for 24 and 48 h,

respectively. Cell suspensions were diluted 1:10 in either fresh

FAB or artificial saliva (mucin, 35 g/L; xylitol, 20 g/L; potassium

chloride, 1.2 g/L; sodium chloride, 0.85 g/L; magnesium chlo-

ride hexahydrate, 0.05 g/L; calcium chloride hexahydrate,

0.2 g/L and dipotassium phosphate, 0.35 g/L14); and 100 mL

added to the wells of a microtitre plate for generation of S.

mutans (24 h) and P. gingivalis (48 h) biofilms and then treated

with 0, 2, 6 or 10% OligoG triclosan. Triclosan is typicallyemployed at a concentration of 0.3% in commercially available

toothpastes and mouthwashes.15 Here, triclosan was applied

to S. mutans and P. gingivalis biofilms at concentrations of 0.3

and 0.01% respectively (0.01% having been determined as the

highest concentration not toxic to P. gingivalis). Dilutions were

carried out in PBS, using 3% isopropanol and PBS alone as

controls. Experiments were performed anaerobically and in

5% CO2 (S. mutans only). Treated biofilms were incubated with

antimicrobial agents for 10 min, 1, 2, 3 or 4 h prior to being re-

suspended and serially diluted (n = 3). Counts were performed

in triplicate on FAA.

2.4. Bacterial attachment to titanium andpolymethylmethacrylate (PMMA) following antimicrobialtreatment

Unpolished titanium (3 mm 11.5 mm; Goodfellow Cam-bridge Ltd., UK) and 1 cm diameter PMMA discs were sterilised

and immersed (24 h, 37 8C, 70 rpm) in 500 mL of 0, 2, 6 or 10%

OligoG 0.3% triclosan, with 3% isopropanol and 0.2%chlorhexidine (CHX) as controls.

Bacterial suspensions of S. mutans or P. gingivalis incubated

anaerobically (for 24 h or 48 h, respectively) were centrifuged

at 3500 rpm for 10 min and re-suspended in 10 mL PBS. This

washing step was repeated twice. On final re-suspension,

the bacteria were diluted to an absorbance of 0.4 (600 nm;

6 108 CFU/mL). Antimicrobial-treated discs were washedin 2 mL PBS and then transferred to a fresh 24-well plate and

incubated with 500 mL of diluted bacterial suspension (2 h,

37 8C, 70 rpm) under anaerobic conditions. Discs were then

washed in 500 mL PBS to remove non-adherent bacteria and

immersed in 1 mL fresh PBS. Discs were vortexed vigorously

for 30 s and the resultant bacterial suspension serially diluted

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 98942.5. Effect of OligoG on biofilm formation

S. mutans and P. gingivalis were grown anaerobically in FAB for

24 h and 48 h, respectively and 35 mL added to the wells of a

sterile 8-well chamber gasket containing 350 mL of 0, 2, 6 and

10% OligoG in FAB (n = 2). These were incubated anaerobically

(24 h, 37 8C). Supernatants were removed and biofilms on the

slide visualised by addition of 5 mL BacLightTM LIVE/DEAD1

stain (Invitrogen) using UV microscopy.

Fig. 1 Bacterial counts (CFU/mL) for (A) S. mutans DSM 20523 an

or microaerophilically (in 5% CO2; S. mutans only) in artificial sa

0.3% triclosan or 0.01% triclosan (Tcs; S. mutans and P. gingivali2.6. Atomic force microscopy (AFM)

Bacterial cultures (48 h) were washed twice (5500 g, 3 min)before mixing with 0.5% OligoG for 20 min. Excess OligoG was

then removed (2500 g, 6 min) before resuspending the cellpellet in dH2O and drying on 0.01% poly-L-lysine coated mica

slides for imaging. A Dimension 3100 AFM (Bruker) was used,

using tapping mode operation in air (0.8 Hz scan speed and

image resolution of 1024).

d (B) P. gingivalis NCTC 11834 biofilms grown anaerobically

liva or fastidious anaerobe broth and treated for 4 h with

s respectively) and/or 0, 2, 6 or 10% OligoG (G) (n = 3).

s D

2,

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 9 8952.7. Statistics

Statistical analysis was performed using Graph Pad statistical

software and one-way analysis of variance (ANOVA) or Tukey

Kramers test in conjunction with ANOVA to compare the

means16 ( p < 0.05 was considered significant).

Fig. 2 BacLightTM LIVE/DEADW viability staining of S. mutan

24 h growth in fastidious anaerobe broth (FAB) containing 0,

red fluorescence indicates cell death.3. Results

3.1. Cytotoxic effects on oral biofilms

Although S. mutans biofilms retained viability after 2 h

incubation with 0.3% triclosan, dilution experiments

revealed P. gingivalis biofilms were more sensitive to

triclosan, with an antimicrobial effect evident at all

concentrations >0.01% (data not shown). In subsequent

experiments, conventional therapeutic concentrations of

triclosan (0.3%) were employed for S. mutans biofilms, whilst

studies on P. gingivalis biofilms employed 0.01% triclosan.

PBS and isopropanol controls produced a straight line

graph between 107 and 108 CFU/mL in all cases (data not

shown).

Neither triclosan nor OligoG alone, or in combination,

exhibited significant anti-biofilm effects ( p > 0.05) against S.

mutans at short exposure times ( 0.05). How-

ever, OligoG potentiated the effect of triclosan against

biofilms established in artificial saliva with addition ofOligoG (10%) shown to induce a significant decrease in S.

mutans bacterial counts under both anaerobic ( p < 0.05) and

microaerophilic ( p < 0.05) growth conditions after incuba-

tion for 1 h.P. gingivalis biofilms grown in artificial saliva showed no

response to OligoG or triclosan alone and no potentiating

effect was seen when used in combination (Fig. 1). P.

gingivalis biofilms grown in FAB appeared to be most

SM 20523 and P. gingivalis NCTC 11834 biofilms following

6 or 10% OligoG. Green fluorescence indicates viable cells;sensitive to triclosan with 10% OligoG, although all treat-

ments showed a trend of decreasing (but not statistically

significant) bacterial number with increased incubation

time.

3.2. Bacterial imaging

Fluorescent microscopy and AFM of bacterial biofilms

established in OligoG showed distinct differences in cell

morphology (Figs. 2 and 3, respectively) with the effect on

bacterial biofilm development varying between both species

studied. S. mutans grown at low OligoG concentrations (2%)

produced distinct chains similar to the control. LIVE/DEAD1

staining demonstrated that, whilst composed of principally

viable cells, their morphology on exposure to higher con-

centrations of OligoG (6 and 10%) was considerably altered

with obvious cellular aggregation and clumping. AFM also

showed clumping of S. mutans chains in the presence of

OligoG.

Surprisingly, staining of P. gingivalis grown in 2% OligoG

showed a marked increase in fluorescent intensity compared

to the FAB control, indicating an increase in biofilm numbers

and growth. In contrast, increasing OligoG concentrations

(6%) resulted in a discernible decrease in cell numbers, andan increase in non-viable cells. AFM showed that the presence

of OligoG induced structural, cellular change with flattening

of the cells.

Using a lower concentration of triclosan (0.01%) showed no

discernible effect on P. gingivalis biofilms after 4 h, either alone

or in combination with 2 or 6% OligoG. Higher concentrations

of OligoG (10%) with 0.01% triclosan were only slightly

effective against P. gingivalis biofilms grown in FAB (and not

statistically significant).

The ability of OligoG to prevent biofilm formation was also

investigated. OligoG (6%) resulted in dramatic decreases inbacterial numbers of P. gingivalis with very sparse biofilm

formation evident; LIVE/DEAD1 staining (and AFM) indicating

that higher OligoG concentrations were also biocidal to

Fig. 3 AFM images of (A) untreated S. mutans (5 mm); (B) S.

mutans + OligoG (20 mm); (C) untreated P. gingivalis (5 mm);

(D) P. gingivalis + OligoG (10 mm).

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 98963.3. Inhibition of bacterial attachment

Bacterial adherence was dependent upon the material

substrate and the bacterial species studied (Fig. 4). P. gingivalis

attachment to treated titanium discs was not significantly

inhibited with triclosan alone ( p > 0.05), but was when

triclosan was used in combination with 2% OligoG(p < 0.01). In contrast, attachment of S. mutans to titanium

was strongly inhibited by triclosan alone ( p < 0.001). Using

OligoG in combination with triclosan showed a further

reduction in attachment, although this was not significant

compared to the triclosan control.

Treatment of PMMA discs with combinations of triclosan

and OligoG had no effect on S. mutans attachment. Interest-

ingly whilst P. gingivalis attachment to PMMA was unaffected

by triclosan alone, a significant decrease was observed when

triclosan was used in combination with 10% OligoG ( p < 0.05)

(Fig. 4).

4. Discussion

Both S. mutans and P. gingivalis are important oral pathogens

which colonise the mouth as biofilms.17,18 Biofilms offer

protection to bacteria, making it more difficult to displace

them from the colonised surface and reducing the efficacy of

almost all antimicrobials.19,20 Although a wide range of

antibacterial dentifrices are available,21 they have limited

antibiofilm efficacy. Hence, we examined the potential role of

the alginate oligosaccharide OligoG, as a non-toxic antibiofilm

adjuvant in dental care.

Triclosan is widely used in toothpastes and

mouthwashes and has been shown to be effective against

oral bacterial biofilms.20 However, a number of criticisms

regarding its widespread use exist, including safety and

environmental concerns. Questions remain regarding its

metabolism in the human body following long term use,

including chronic dermal toxicity and carcinogenicity. In

addition, triclosan can build up in the environment

resulting in marine toxicity.22 There have consequently

been calls for a reduction in the concentrations employed in

commercially available healthcare products. OligoG has

been shown to effectively potentiate the effect of antibac-

terial agents in vitro against a number of bacterial patho-

gens.8 We have shown here that this potentiation may also

be observed for the action of triclosan in vitro with S. mutans

suggesting that lower concentrations of triclosan, used in

combination with OligoG, could achieve similar antibacteri-

al results as those observed at currently employed com-

mercial concentrations. In particular, S. mutans biofilms in

artificial saliva, showed a marked decrease in viability

following exposure to a combination of OligoG and triclosan

compared to triclosan alone. This data supports the

hypothesis that OligoG may be beneficial in a clinical

setting, where bacteria do not grow in nutrient-rich media,

but rather as dental plaque biofilms, under nutrient-

limited conditions.

Interestingly, P. gingivalis biofilms were more sensitive totriclosan than those of S. mutans, with 0.3% triclosan

completely eradicating these biofilms within 2 h of exposure.planktonic P. gingivalis. S. mutans showed a similar, although

less pronounced pattern typified by less marked cell death.

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 9 8970

1

2

3

4

5

6

7

8M

ean

Log 1

0 CFU

/mL

S. m

utan

s TITANIUM

**Although still viable, the resultant S. mutans biofilm appeared

as small, compact, dense areas of growth or aggregates.

Under starvation conditions S. mutans cells become shrunken

and distorted when stressed23 suggesting that they may use

aggregation as a survival mechanism.24 Such morphological

changes appear to have resulted in smaller, denser biofilms,

suggesting it was injurious to bacterial growth and survival.

Further cell death and morphological changes may also be

observed at longer incubation times. Alginates have been

shown to disrupt intermolecular interactions in extracellular

polymeric substances such as mucus,25 reflecting reduced

cross-linking and weakening of the biofilm structure.

The cytotoxicity results in artificial saliva for mature P.

gingivalis biofilms appear at odds with the LIVE/DEAD1

staining, which clearly showed reduced bacterial viability at

6% OligoG. Whilst triclosan acts as a biocide, at lowerconcentrations, it is bacteriostatic, targeting fatty acid

Isop ropano l Triclosan Tcs + 2%G Tcs + 6%G Tcs +10 % G CHX

Treat ment

Isop ropano l Triclosan Tcs + 2%G Tcs + 6%G Tcs +10 %G CHX0

1

2

3

4

5

6

7

8

Mea

n Lo

g 10 C

FU/m

L

Treat ment

P. g

ingi

valis

*

Fig. 4 Bacterial counts (CFU/mL) for S. mutans DSM 20523 and

polymethylmethacrylate (PMMA) or titanium. Surfaces were trea

chlorhexidine (CHX) (n = 3) (*p < 0.05; **p < 0.001).POLYMETHYLMETHACRYL ATE

0

1

2

3

4

5

6

7

8

Mea

n Lo

g 10 C

FU/m

Lsynthesis. The nutrient-limited environment of the artificial

saliva may have resulted in characteristically low levels of

metabolic activity, where cells often enter a dormant viable

but not culturable state under nutrient-deprived conditions,

which paradoxically affords increased resistance to antimi-

crobials26 and may explain the increased resistance observed

in this instance.

Many studies have focused on the surface modification of

dental implants to reduce bacterial attachment.2729 It has also

previously been shown that dentifrices containing triclosan

were able to reduce bacterial numbers on titanium implants.30

In this study, OligoG was combined with triclosan to prevent

bacterial attachment to titanium surfaces and biofilm forma-

tion. A significant decrease in P. gingivalis attachment to the

titanium surface was seen following treatment with triclosan

combined with low OligoG (2%) concentrations compared to

triclosan or chlorhexidine alone. Thus use of OligoG in the

Isop ropano l Triclosan Tcs + 2%G Tcs + 6%G Tcs + 10%G CHX0

1

2

3

4

5

6

7

8

Mea

n Lo

g 10 C

FU/m

L

Treat ment

*

Isop ropano l Triclosan Tcs + 2%G Tcs + 6%G Tcs + 10%G CHX

Treat ment

P. gingivalis NCTC 11834 attachment to

ted for 18 h with triclosan (Tcs) W0, 2, 6 or 10% OligoG (G) or

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 8 9 2 8 9 9898treatment of exposed dental implants and peri-implantitis

cases; providing a biodegradable, anti-biofilm effect at these

sites, without chemically altering the surface of the implant,

warrants further investigation.

PMMA is a major component of removable dentures which

are prone to colonisation by bacteria and fungi. Inefficient oral

and denture care is common amongst denture wearers.31

Denture pastes can be abrasive, causing roughening of the

denture surface, and thereby facilitating bacterial attach-

ment.32 Immersion soaks may therefore be favoured, particu-

larly if the active antimicrobial can be shown to remain active

following rinsing. Immersing PMMA in triclosan alone or in

combination with 2, 6 or 10% OligoG had no effect on

subsequent attachment of S. mutans. However, P. gingivalis

numbers recovered from PMMA discs treated with triclosan

and 10% OligoG were significantly reduced. Interestingly, the

prototypical antimicrobial agent chlorhexidine was less

effective at reducing bacterial attachment than expected,

with no significant difference to the PBS control being

observed in this model.

These studies showed that the novel, safe antimicrobial

OligoG, alone or in combination therapy, can potentiate the

activity of existing antimicrobials such as triclosan against

oral biofilms. OligoG may also have a role in periodontal

disease and peri-implantitis by preventing attachment and

infection of the implant surface by P. gingivalis. A potential

environmental benefit could also be realised by decreasing the

concentrations of triclosan currently used in clinical products

if used in conjunction with OligoG.

Acknowledgements

This study was supported by Algipharma AS, Sandvika,

Norway and the Faculty of Dental Surgery, Royal College of

Surgeons, England (C.E).

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An invitro study of alginate oligomer therapies on oral biofilmsIntroductionMaterials and methodsChemicals and reagentsBacteriaExposure of oral biofilms to OligoG and triclosanBacterial attachment to titanium and polymethylmethacrylate (PMMA) following antimicrobial treatmentEffect of OligoG on biofilm formationAtomic force microscopy (AFM)Statistics

ResultsCytotoxic effects on oral biofilmsBacterial imagingInhibition of bacterial attachment

DiscussionAcknowledgementsReferences