Basic Research—Technology

Antibacterial and Anti-biofilm Activity of AH Pluswith Chlorhexidine and CetrimideM. Estela Bail�on-S�anchez, DDS,* Pilar Baca, DDS, MD, PhD,† Matilde Ruiz-Linares, DDS, PhD,‡

and Carmen Mar�ıa Ferrer-Luque, DDS, MD, PhD§

Abstract

Introduction: The use of root canal filling materialswith antibacterial activity can be considered beneficialto reduce the remaining microorganisms in the root ca-nal system, where Enterococcus faecalis is oftenfound, and prevent recurrent infection. The aim of thisstudy was to evaluate the antimicrobial activity and ca-pacity for inhibiting E. faecalis biofilm formation of AHPlus, alone and mixed with chlorhexidine (CHX),cetrimide (CTR), and combinations of the two.Methods: AH Plus alone and mixed with 1% and 2%CHX, 0.1%–0.5% CTR, and combinations of bothwere tested to assess antimicrobial activity by amodified direct contact test and determine inhibitionof E. faecalis biofilm formation at 24 hours. The resultswere expressed as log10 viable counts. Eradication andinhibition of biofilm formation were understood as nobacterial growth or log10 reduction = 5 with respect tothe control (AH Plus alone). Results: AH Plus + CHXshowed a low antimicrobial activity with respect tothe control (at 2%, log10 reduction = 1.30). None ofthe tested concentrations achieved eradication orinhibition of biofilm. AH Plus + CTR showed adirect relationship of concentration-antimicrobial effect,reaching a log10 reduction of 2.92 at 0.5% and inhibitionof biofilm formation at 0.2%. With the combinationCHX + CTR, lower concentrations were needed for thesame effect, and eradication and inhibition of biofilmwere achieved. Conclusions: The addition of CHX,CTR, or some combination of both to AH Plus confersit with bactericidal and anti-biofilm activity againstE. faecalis. (J Endod 2014;40:977–981)

Key WordsAH Plus, biofilm, cetrimide, chlorhexidine, direct contacttest, Enterococcus faecalis

From the *University of Granada, Campus de Cartuja, Colegio Mde Cartuja, Colegio M�aximo s/n; ‡Department of Paediatric Denti§Department of Dental Pathology and Therapeutics, School of Dent

Address requests for reprints to Dr Matilde Ruiz-Linares, DepartmSpain. E-mail address: matr@ugr.es0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.11.020

JOE — Volume 40, Number 7, July 2014

The most important objectives of endodontic treatment are the elimination ofmicroorganisms from the root canal system and the prevention of subsequent

reinfection. However, in some cases the complete elimination of bacteria fromthe root canal system is impossible (1). The use of root canal filling materials withantibacterial activity can reduce the number of remaining microorganisms (2, 3),prevent recurrent root canal infection, and aid the repair process of apical andperiapical tissues (4). Enterococcus faecalis is often found in asymptomaticfailing root canal–treated teeth, in primary necrotic cases, and in persistent apicalperiodontitis (5, 6). When grown as a biofilm in the root canal, these bacteriapenetrate into dentinal tubules and resist nutritional deprivation (5).

A great variety of endodontic sealers are available commercially; most of them arebased on zinc oxide–eugenol, epoxy resin, calcium hydroxide, and glass ionomers (7).AH Plus (Dentsply DeTrey, Konstanz, Germany) is an epoxy resin–based root canalsealer with good physical properties, good adhesion to dentin, and excellent fluidityand stability in aqueous solution (8). Aside from being biocompatible (9, 10), it isnot genotoxic (11) and exhibits good tissue tolerance, sealing ability, and long-termdimensional stability (12). Its antimicrobial properties have been shown to be activeagainst Staphylococcus aureus, Escherichia coli, Streptococcus mutans, andStaphylococcus epidermidis (4).

Chlorhexidine (CHX) is a potent antimicrobial frequently used in endodontics(13, 14). Vianna et al (15) report that it is able to inactivate many endodontic-resistant organisms in as little as 15 seconds of contact time. Cetrimide (CTR) is acationic surfactant that has demonstrated its ability to eradicate E. faecalis biofilmin vitro (14) and ex vivo (16, 17).

In the last decade, antimicrobial agents have been incorporated into dentalmaterials to lend them antimicrobial activity (18, 19). The incorporation of CHXand/or CTR into glass ionomer cement (GIC) (20–22) is known to confer it withbeneficial antibacterial properties. It was recently shown that the incorporation ofbenzalkonium chloride and cetylpyridinium chloride into endodontic cementsimproves their antibacterial effect on S. mutans, Lactobacillus casei, andActinomyces viscosus (23). In previous research, the authors of the present studyshowed that the addition of 1% and 2% CHX, 0.1% up to 0.5% CTR, or combinationsof the two does not significantly alter some important physical properties of AH Plus(24). To evaluate the antimicrobial effects of endodontic sealers and pastes, the agardiffusion test is widely used (4, 22, 23). Because this technique presents severallimitations, Weiss et al (25) described a direct contact test (DCT) assay designed toovercome them. The DCT quantitatively assesses the effect of direct contact betweenorganisms and materials (26). Because bacteria in the root canal grow as biofilm

�aximo s/n; †Department of Preventive Dentistry, School of Dentistry, University of Granada, Campusstry, School of Dentistry, University of Granada, Campus de Cartuja, Colegio M�aximo s/n; andistry, University of Granada, Campus de Cartuja, Colegio M�aximo s/n, Granada, Spain.ent of Stomatology, School of Dentistry, Campus de Cartuja, ColegioM�aximo s/n, E-18071 Granada,

Antibacterial Activity of AH Plus 977

TABLE 2. Viable Counts of E. faecalis Biofilm Formed onto AH Plus Combinedwith CHX and/or CTR

CTR

CHX

None 1% 2%

None 4.78 (0.06)*,a 4.55 (0.09)b 3.18 (0.07)c

0.1% 1.95 (0.05)d 0 00.2% 0 0 00.3% 0 0 00.4% 0 0 00.5% 0 0 0

Log10 CFU + 1/mL. Mean (standard deviation).

The same superscript letters show differences not statistically significant by Duncan test.

*Control: AH Plus without CHX and CTR.

Basic Research—Technology

(27), particularly E. faecalis, it is necessary to determine the antimicro-bial activity of materials that are able to inhibit its formation. Therefore,the aim of this study was to evaluate the antimicrobial activity andcapacity of inhibiting E. faecalis biofilm formation of the AH Plus sealermixed with either CHX or CTR and combined with both.

Materials and MethodsIn both tests, antimicrobial activity and biofilm formation, AH Plus

was used alone as the control group and combined with CHXdigluconate (liquid, 1% and 2%) (Guinama, Alboraya, Spain), CTR(powder, 0.1%–0.5%) (Sigma-Aldrich, Steinheim, Germany), andboth (concentrations of combinations are shown in Tables 1 and 2).Concentrations of 10% and 20% CHX digluconate were combinedwith AH Plus in a ratio 1/10 (v/v). CTR powder was added to AH Plusin ratios ranging from 1/100–1/20 (w/w) to obtain concentrationsfrom 0.1%–0.5%. Combinations of both antimicrobials were preparedwith the different concentrations of CHX and CTR (v/w) and were addedin the same ratios to AH Plus as described above. All these procedureswere performed under sterile conditions in a laminar flow chamber.In both tests the materials were left to set for 48 hours (24) in sterileconditions.

The bacterial suspensions were prepared following the samemethodology for all experiments. From a subculture of E. faecalisATCC 29212, colonies were suspended in brain-heart infusion (BHI)broth to match the optical density of the 1.0 McFarland standard(3 � 108 colony-forming units [CFU] per mL), determined by usinga calibrated turbidimeter. This suspension was diluted 30-fold inbroth to obtain an initial bacterial suspension of approximately1 � 107 CFU/mL to be used for DCT and biofilm determination. Theexperiments were performed under sterile conditions in a laminarflow chamber (model Bio-II-B; Telstar S.A., Terrasa, Spain).

Antimicrobial Activity TestTo test the antimicrobial activity of materials, we used a method-

ology based on the DCT (25) adapted by Zhang et al (2). A 96-wellmicrotiter plate was used. To standardize the area of material tested,a customized silicon mold was made to fit inside each well in the plate,leaving a small area on one side to allow us to introduce the materialalways in equal amounts. The materials were introduced by using asterile syringe-needle system (BD DISCARDIT II; Becton DickinsonS.A., Fraga, Huesca, Spain) in the space between the well andcustomized silicon mold, and the material was left to set. Thereafter,the molds were removed from each well, and the 96-well microtiterplate was held vertically so that 10 mL of the bacterial suspension ofE. faecalis could be carefully placed over thematerials. After incubationat 37�C for 1 hour in a humid atmosphere, ensuring contact betweenthe bacterium and the material, we turned the plate to a horizontal

TABLE 1. Viable E. faecalis Counts by AH Plus Combined with CHX and/or CTRby Using the Modified DCT

CTR

CHX

None 1% 2%

None 6.28 (0.06)*,a 5.30 (0.13)b 5.23 (0.28)b

0.1% 4.65 (0.03)c 4.52 (0.06)d,e 4.56 (0.04)c,d

0.2% 4.51 (0.06)d,e 4.49 (0.02)d,e 4.45 (0.06)d,e,f,g

0.3% 4.42 (0.08)e,f,g 4.39 (0.05)e,f,g 00.4% 4.35 (0.08)f,g,h 4.26 (0.09)h 00.5% 3.24 (0.26)i 3.04 (0.88)j 0

The same superscript letters show differences not statistically significant by Duncan test.

*Control: AH Plus without CHX and CTR.

978 Bail�on-S�anchez et al.

position; then 220 mL BHI was added to each well and gently mixedwith the pipette for 1 minute. Finally, 50 mL was taken from eachwell, and serial dilutions were performed and plated for viable cellcounting. Each material was tested twice, and the procedure wasperformed in duplicate (n = 4/group). Four wells with AH Plus aloneand noninfected were included as negative controls.

Biofilm Formation on Material TestedTo study E. faecalis biofilm formation, we used a methodology

based on the Calgary biofilm device (commercially available as theMBEC–high throughput [HTP]; Innovotech, Edmonton, Alberta,Canada), originally described by Ceri et al (28) and adapted andmodified for E. faecalis (6) and the materials tested.

The top half of the MBEC-HTP is a lid with 96 identical pegs, inthis case used to make the silicon molds, which were sterilized for45 minutes in hydrogen peroxide gas plasma (Sterrad-50; AdvancedSterilization Products, Johnson & Johnson, Irvine, CA). Each materialwas introduced in the molds to obtain identical cylinders. Thedimensions of each cylinder were 6 mm (height) by 3.90 mm(maximum diameter). The amount of sealer in each cylinder wasapproximately 0.065 mL. A sterile orthodontic wire 1 cm in lengthwas placed in each cylinder while the material set, always leavingapproximately 3 mm of it outside the material to facilitate manipulation(Fig. 1).

The 96-well microtiter plate was inoculated with 180 mL/well ofthe suspension of E. faecalis in BHI, after which the cylinders ofeach material were introduced. The microtiter plate-cylinder set wasthen placed on a rocking table (model Swing Sw 8 10000-00015;OVAN, Badalona, Spain) and incubated at 37�C for 24 hours at 5 rocksper minute. Biofilms forming onto the cylinders were rinsed twice byplacing the cylinders in wells with 200 mL 0.9% saline solution for2 minutes (each time) and then transferring them to a 96-wellmicrotiter recovery plate with 200 mL BHI/well, which was sonicatedin a water-table ultrasonic bath (model 5510E–MT; Branson, Danbury,CT). Ten minutes of ultrasonification were sufficient time to recoverbiofilm (with no significant differences versus 20 minutes) withoutaffecting the cylinders’ integrity (height/diameter), which was alsodetermined by the optical density of the broth after subjecting theuninfected cylinders to ultrasonication for 10 minutes.

Each material was tested twice, and the procedure was performedin duplicate (n = 4/group). Four cylinders of AH Plus alone andnon-infected were included as negative controls. The disrupted biofilmswere diluted serially and plated for viable cell counting; data weretransformed to a logarithmic scale.

Results of antimicrobial activity of the tested materials andE. faecalis biofilm formation onto them were respectively expressed

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Figure 1. Silicon molds and cylinders of material to test inhibition of biofilm formation.

Basic Research—Technology

as log10 CFU + 1/mL. Eradication and inhibition of biofilm formationwere understood as no bacterial growth or a log10 reduction = 5with respect to the control (AH Plus alone). Data were analyzed by using1-way analysis of variance followed by the Duncan post hoc test tocompare among groups.

ResultsAll negative controls performed throughout the experiment

showed no bacterial growth. The overall results of antimicrobial activity

Figure 2. Antimicrobial activity expressed as log10 reduction viable counts, with re(A), CTR (B), or both (C and D).

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and the inhibition of E. faecalis biofilm formation are shown in Tables 1and 2, respectively, which include the values of the controls (AH Plusalone). Figure 2 displays the antimicrobial activity as the log10 reductionin viable counts with respect to the control of all materials tested.

Regarding antimicrobial activity by DCT, AH Plus mixed onlywith 1% and 2% CHX showed lower antimicrobial activity (respectivelog10 viable counts of 5.30 and 5.23) than the control (log10 6.28).The combinations of AH Plus with CTR showed a concentration-dependent antimicrobial activity, although none of the testedconcentrations achieved eradication (log reduction = 5). CTR in the

spect to the control (log10 CFU/mL = 6.28� 0.06), of AH Plus mixed with CHX

Antibacterial Activity of AH Plus 979

Basic Research—Technology

highest concentration (0.5%) obtained a 2.92 log10 reduction withrespect to the control of AH Plus alone.

The inhibition of biofilm formation obtained by AH Plus combinedwith CHX showed a direct relationship with the concentration used, butno full inhibition. However, CTR inhibited E. faecalis biofilm formationat a concentration of 0.2%.

The results of the combinations of both antimicrobials mixed withAH Plus indicate a synergic antimicrobial effect seen in the antimicrobialactivity as well as in the inhibition of biofilm formation. Thus, 0.3% CTRwas necessary to eradicate E. faecalis when combined with 2% CHX,and only 0.1% CTR attained inhibition of biofilm formation when mixedwith any CHX concentration.

DiscussionAlthough chemomechanical preparation is essential for elimi-

nating bacteria from the root canal system (2), an antibacterial effectof root canal filling materials is helpful, because if bacteria remain indentinal tubules, they may become reservoirs for reinfection (29).Endodontic cements are essential to ensure proper sealing of rootcanals, and among their other properties, it would be desirable thatthey exerted antimicrobial activity on contact with microbes andbiofilms, ideally maintaining this effect over time (2).

To assay antimicrobial activity, we used a modification of the DCT(25), which provides for a smooth and standardized surface, givingoptimal exposure to the bacteria. To test the inhibition of biofilmformation in this study, a system based on the Calgary device (28)was developed, but replacing the original polystyrene peg-lids withidentical cement peg-lids for testing. Silicon molds from the impressionof peg-lids allowed us to obtain specimens of the same size, shape, andsurface smoothness. Such standardization of the techniques used isconsidered a critical step for any biofilm growth in an in vitro orin vivo model system (30) involving comparisons.

AH Plus, an epoxy resin–based root canal sealer, was selectedbecause several in vitro (2, 4, 31), in vivo, and clinical studies(32) indicate its suitability for endodontic therapy. CHX and CTR areeffective antimicrobial agents that have been incorporated successfullyin GICs (20, 22, 33). Furthermore, it was proven that the incorporationof 2 antimicrobials belonging to the quaternary ammonium group, suchas CTR, increase the antimicrobial effect of several endodontic sealers(23), although these authors assayed cariogenic bacteria by the agardiffusion test and recommended to determine their physical properties.In this sense, it has been recently demonstrated that adding CHX, CTR,or both to AH Plus at any of the combinations tested in this study doesnot significantly alter setting time, flow, solubility, and radiopacity (24).The results remained consistently within the American NationalStandards Institute/American Dental Association requirement for end-odontic sealers (34). However, further characterization experimentsconcerning the dimensional stability, adhesion to dentin/gutta-percha, and leakage of the sealer/material set should be undertakenbefore these combinations can be considered for clinical application.

Overall, these results are consistent with previous studies (2, 3,35) and show that AH Plus alone was not capable of killing bacteriaor inhibiting E. faecalis biofilm formation. However, the addition ofCHX, CTR, or CHX + CTR conferred antimicrobial properties to thissealer cement, as observed in both tests; besides a direct relationshipbetween dose and effect, we confirmed that the combinations of CHXand CTR ensured better results than their single use (17).

AH Plus-CHX at 1% and 2% showed a low antimicrobial activitywith respect to the control, with respective values of log10 5.30 and5.23 and significant differences with respect to AH Plus alone(log10 6.28). From a microbiological point of view this would be

980 Bail�on-S�anchez et al.

irrelevant, because log10 reductions of 0.9 and 1.03 are very far fromthe reduction of 5 considered eradication. The low antimicrobialactivity, similar for both concentrations, could be attributed to thefact that CHX digluconate acts as a cross-linking agent (36), whichwould be related to its low antimicrobial activity. Regarding theinhibition of biofilm formation, the results point to a limited effect.

AH Plus + 0.5% CTR achieved a log10 reduction of 2.92; however,a concentration of 0.2% CTR sufficed to inhibit biofilm formation. Thisresult is logical, because a greater concentration of antimicrobial agentis needed for a bactericidal effect than for inhibiting properties such asbiofilm formation (37).

In the past decade, antimicrobial agents have been added to dentinadhesives and dental materials to enhance antimicrobial activity (8, 19).The mixture of AH Plus with CHX + CTR was seen to improve theantimicrobial effect. By using a DCT, 2% CHX combined with 0.3%CTR achieved E. faecalis eradication with a log10 reduction >5 withrespect to control, and 1% CHX + 0.1% CTR was able to inhibitbiofilm formation completely. This enhanced capacity has beendemonstrated previously in irrigating solutions (14, 17). Moreover,lower concentrations of the antimicrobial agents are sufficient to attainthe same results. Previous studies of GICs (21, 22) with incorporationof CHX and CTR have shown that the antimicrobial properties areaccompanied by a deterioration of physical properties. This potentialis dose-dependent, however; 1% CTR does not exert this effect, asopposed to the concentration of 2.5% (22). In this regard, it is essentialto ensure that the concentrations of antiseptics added to AH Plus areeffective as antimicrobials but do not alter their physical properties.

ConclusionThe DCT and biofilm analysis method used here show that the

addition of CHX, CTR, or both to AH Plus proves beneficial, increasingthe bactericidal and anti-biofilm capacity of this material. However,these are preliminary tests, and further ex vivo experiments in rootcanals are needed to confirm these results in vitro.

AcknowledgmentsThe authors thank Francisca Castillo P�erez and Gertrudis

G�omez Villaescusa for their technical assistance. This study wassupported by the Research Group CTS-167 of the Junta deAndaluc�ıa, Spain.

The authors deny any conflicts of interest related to this study.

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