Antibacterial efficacy of endodontic irrigating solutions andtheir combinations in root canals contaminated withEnterococcus faecalis

Renata Dornelles-Morgental, MSc,a Juliane Maria Guerreiro-Tanomaru, PhD,b

Norberto Batista de Faria-Júnior, MSc,a Marco Antonio Hungaro-Duarte, PhD,c

Milton Carlos Kuga, PhD,b and Mário Tanomaru-Filho, PhD,b Araraquara and Bauru, SP, BrazilSÃO PAULO STATE UNIVERSITY AND UNIVERSITY OF SÃO PAULO

Objective. The objective of this study was to evaluate the antibacterial efficacy of irrigating solutions and theircombinations against Enterococcus faecalis.Study design. One hundred ten single-rooted human teeth were inoculated with E. faecalis and incubated for 21 days.Teeth were divided according to the irrigant: Group I (GI), 2.5% sodium hypochlorite solution (NaOCl); GII, 2.5%NaOCl � 10% citric acid; GIII, 2.5% NaOCl � apple cider vinegar; GIV, apple cider vinegar; GV, 2% chlorhexidinesolution; GVI, 1% peracetic acid; GVII, saline solution. Microbiological samples were taken after root canalpreparation and 7 days later. Data were submitted to ANOVA (5%).Results. All solutions promoted reduction of E. faecalis after instrumentation, but bacterial counts were higher in thefinal sample. GI, GV, and GVI had lower bacterial counts than the other groups.Conclusions. The irrigating solutions may present activity but do not eradicate E. faecalis in the root canal system.

(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;xx:xxx)

Microorganisms and their products play an essential rolein the development of pulpal and periapical patholo-gies.1-3 Culture and molecular techniques allowed thedetection of more than 460 species of bacteria in differenttypes of endodontic infections.4,5 Enterococcus faecalis isa gram-positive facultative anaerobe capable of invadingthe dentin tubules6 and is resistant to several irrigatingsolutions and medications used in endodontics.7

Sodium hypochlorite (NaOCl) is the most commonlyused irrigating agent because of its broad microbicidalactivity and tissue-dissolving capability.8,9 However,variables, such as concentration,10 temperature,11 andpH,12,13 can affect its efficacy. At lower pH, the chlo-rine available in the NaOCl solution presents as hypo-chlorous acid (HOCl), which is more active than thehypochlorite anion (OCl) that prevails at higher pH.14,15

This study was supported by FAPESP—Foundation for the Supportof Research, State of São Paulo, Brazil (Grant 2008/03298-2).aGraduate Student, Araraquara Dental School, UNESP, São PauloState University, Department of Restorative Dentistry, Araraquara,SP, Brazil.bProfessor, Araraquara Dental School, UNESP, São Paulo State Uni-versity, Department of Restorative Dentistry, Araraquara, SP, Brazil.cProfessor, Bauru Dental School, USP, University of São Paulo,Department of Restorative Dentistry, Bauru, SP, Brazil.Received for publication Dec 4, 2010; accepted for publication Feb 3,2011.1079-2104/$ - see front matter© 2011 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2011.02.004

Therefore, the combination of NaOCl with acidic solu-tions, despite affecting its stability, may increase itsantimicrobial effect.12

Chlorhexidine is a potent antiseptic, used as an end-odontic irrigant, presenting a wide spectrum of antimi-crobial action and substantivity.16-18 Its main disadvan-tage is the lack of ability to dissolve organic matter,19

which negatively affects its cleaning capacity.20

Alternative irrigating solutions demonstrating anti-bacterial action and ability to clean the dentin wallshave been investigated. Apple cider vinegar is activeagainst endodontic microorganisms21 and has the abil-ity to remove the smear layer.22 Peracetic acid is adisinfectant widely used in the food industry and inhospitals.23 Its use in endodontics has been recentlyinvestigated and revealed its ability to remove thesmear layer.24 Because of its excellent antimicrobialaction in the presence of organic matter, this substancehas also been recommended in other applications, suchas disinfection of water lines in dental units25 and ofacrylic resins.26 These properties justify its evaluationfor use as an endodontic irrigant.

A great variety of substances have been evaluated fortheir action against E. faecalis.7,27,28 Moreover, somestudies have suggested the combination of NaOCl withacidic solutions to increase its antibacterial activity.12,13

The present ex vivo study aimed to evaluate the effec-tiveness of different endodontic irrigating solutions and

their combinations against E. faecalis.

1

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MATERIAL AND METHODSStandardization of the specimens

This study was approved by the Research EthicsCommittee of the School of Dentistry of Araraquara—UNESP. One hundred ten extracted single-rooted hu-man teeth with a single straight root canal were se-lected. Crowns were sectioned with an IsoMet 1000precision saw (Buehler, Ltda, Lake Bluff, IL) to stan-dardize the length of each specimen at 15 mm.

A #3 Gates-Glidden drill (Dentsply-Maillefer, Bal-laigues, Switzerland) was used to prepare the root canalorifices (3 mm). The root canals were instrumented 1mm short of the total length up to a #35 Kirschner file(K-file) (Dentsply-Maillefer). Saline solution was usedfor irrigation. Next, the root canals were filled with17% trisodium EDTA (Biodinâmica, Ibiporã, PR, Bra-zil) for 3 minutes, and then washed with 5 mL of salinesolution.

The root apices were then sealed with compositeresin and all external root surfaces were made imper-meable with epoxy adhesive, except for the cervicalaccess. The specimens (n � 15) were randomly dividedinto seven 24-well cell culture microplates (CorningIncorporated, Corning, NY) and 1 control microplate(n � 5). Specimens were attached to the wells withacrylic resin and sterilized by gamma rays from a cobalt60 source (EMBRARAD, Cotia, SP, Brazil).

Contamination with E. faecalisThe procedures were carried out in a laminar flow

chamber (VecoFlow Ltda, Campinas, SP, Brazil). Stan-dardized suspensions of E. faecalis ATCC 29,212 werespectrophotometrically obtained (1.5 � 108 colony-forming units [CFU]/mL). Tryptic soy broth (TSB;Difco, Detroit, MI) was added to the bacterial suspen-sion at a 1:1 ratio and the root canals were contami-nated with 20 �L of this mixture. A sterile cotton pelletwas soaked with TSB and placed at the root canalentrances.

The microplates containing the specimens were keptin a microaerophillic environment at 37°C for 21 days.

Table I. Experimental groups (irrigants and neutraliziGroups n Irrigan

GI 15 2.5% NaOCl solutioGII 15 2.5% NaOCl � 10%GIII 15 2.5% NaOCl � appGIV 15 Apple cider vinegarGV 15 2% chlorhexidine soGVI 15 1% peracetic acid soGVII 15 0.85% sterile saline

G, group.

Throughout this period, the root canals received TSB

on alternate days by means of a 0.5-mL insulin syringe(Becton Dickinson, Curitiba, PR, Brazil), as describedby Cardoso et al.28

Afterward, samples were harvested from each rootcanal so as to confirm contamination (initial sample)using 3 sterile paper points (Tanari Industrial Ltda, SãoPaulo, SP, Brazil) and transferred to tubes containingsterile saline. Decimal serial dilutions were made andaliquots were seeded in triplicate onto Petri dishescontaining tryptic soy agar (TSA; Difco) and incubatedin microaerophillic conditions at 37°C for 48 hours.Bacterial growth was measured by the CFU/mL countsof E. faecalis.

Division of the experimental groupsSeven experimental groups were established accord-

ing to the irrigant used (Table I). In group II (GII) andGIII, the solutions were mixed in a syringe beforeirrigation. The ratio of each solution was determined bypH analysis: ratios resulting in pH 5 were selected. Atthis pH level, the antimicrobial activity of NaOCl so-lutions is enhanced29 because of the availability of freechlorine as HOCl.14 The specimens in the control groupwere not submitted to biomechanical preparation.

All root canals in the experimental groups wereinstrumented up to a #50 K-file. Next, they were in-strumented up to a #70 K-file following a step-backtechnique. At each instrument change, root canals wereirrigated with 2 mL of the corresponding irrigant. Totalpreparation time was standardized at 10 minutes perspecimen. Finally, 2 mL of a specific neutralizing agentwas used (Table I).

Samples and microbiological analysisSamples were taken immediately after the root canal

instrumentation (postpreparation sample). Decimal di-lutions of the suspensions were plated and incubated,similar to the procedures followed after the initial sam-ple. The root canals were then filled with sterile salineand a sterile cotton pellet was placed at the canalentrance. The microplates containing the specimens

nts)Neutralizing agent

1% Sodium thiosulfateacid 1% Sodium thiosulfate � 1% Tween 80vinegar 1% Sodium thiosulfate � 1% Tween 80

1% Sodium thiosulfate � 1% Tween 800.5% lecithin � 1% Tween 801% sodium thiosulfate � 1% Tween 800.85% sterile saline solution

ng aget

ncitric

le cider

lutionlutionsolution

were covered and incubated once again in a microaero-

OOOOEVolume xx, Number x Dornelles-Morgental et al. 3

phillic environment at 37°C for 7 days, after which anew sample was taken (final sample). Harvesting ofsamples from the control group (canals that were notprepared) was performed at the same times.

Statistical analysisResults were submitted to logarithmic transforma-

tion and analyzed using the GraphPad Prism 3.0 soft-ware (San Diego, CA). Analysis of variance (ANOVA)and Tukey tests were used for comparison between theexperimental groups. For comparison between the sam-ples within each group, repeated measures ANOVAand the Bonferroni tests were used. The significancelevel was set at P � .05.

RESULTSContamination methodology was confirmed by the

recovery of pure E. faecalis strains from all teeth in theinitial sample performed 21 days after incubation,which revealed similar CFU/mL counts for all groups(Table II).

Comparison among GI, GIII, GV, and GVI at thepostpreparation period revealed that these groups hadsimilar results, but had significantly lower counts thanthe other groups (P � .05). At the final period, GI, GV,and GVI presented similar results (P � .05), but dif-fered significantly from the other groups, as shown inTable II.

According to Table III, immediately after biome-chanical preparation, all irrigants reduced the number

Table II. Comparison between the groups in the inCFU/mL log)

Group Initia

GI, 2.5% NaOCl 11.00 (�GII, 2.5% NaOCl � 10% citric acid 10.85 (�GIII, 2.5% NaOCl � apple cider vinegar 11.15 (�GIV, apple cider vinegar 11.13 (�GV, 2% chlorhexidine 10.65 (�GVI, 1% peracetic acid 11.07 (�GVII, saline 11.03 (�

Different superscript letters a,b,c indicate statistically significant diffeCFU, colony-forming units; G, group.

Table III. Comparison between the initial, postpreparatSample GI GII GIII

Initial 11.00a 10.85a 11.15a

Postpreparation 0.00b 1.47b 0.00b

Final 6.40c 8.89c 9.7c

Different letters indicate statistically significant difference accordingCFU, colony-forming units; G, group.

of bacteria inside the root canal. However, at the final

sample, a significant increase in the CFU/mL countswas observed in all groups.

The 5 specimens in the control group, which werenot instrumented after the contamination period, con-firmed the viability of E. faecalis throughout the exper-imental phases.

DISCUSSIONThe experimental model we adopted allows simula-

tion of endodontic infection in the root canal systemand dentin tubules, closely resembling in vivo clinicalsituations.30 E. faecalis was used because of its highprevalence in cases of endodontic treatment failure andalso because of its ability to form biofilm and survive asa monoculture.7 A 21-day period of contamination al-lows diffusion of the E. faecalis suspension through thedentin tubules, as reported by Berber et al.10 and Car-doso et al.28 Seven days after instrumentation, a secondsample was taken to verify the permanence of viablebacteria inside the root canal system.28,30,31

The results of the present study demonstrated thatbiomechanical preparation using all irrigating solutionssignificantly reduced the presence of E. faecalis. How-ever, an increase in the number of CFU/mL 7 days afterinstrumentation was observed in all groups. This maybe because of biofilm formation, rendering the bacterialcells more resistant than when in planktonic form,besides the persistence of bacteria inside the dentintubules and ramifications of the main canal. Theseresults are in agreement with those reported by Byström

ostpreparation, and final samples (mean and SD of

Postpreparation Final

0.00 (�0.00)a 6.40 (�2.75)a

1.47 (�1.45)b 8.89 (�1.69)b

0.00 (�0.00)a 9.71 (�1.21)b

2.06 (�1.78)b 10.47 (�1.28)b,c

0.00 (�0.00)a 7.77 (�1.23)a,b

0.00 (�0.00)a 8.01 (�1.26)a,b

2.06 (�1.78)b 9.68 (�1.30)b

ccording to the Tukey test (P � .05).

d final samples within each group (mean CFU/mL log)IV GV GVI GVII Control

13a 10.65a 11.07a 11.03a 11.51a

06b 0.00b 0.00b 2.06b 11.02a

47a 7.77c 8.01c 9.68c 10.23a

Bonferroni test (P � .05).

itial, p

l

.59)a

.64)a

.80)a

.73)a

.89)a

.56)a

.70)a

rence a

ion, anG

11.2.

10.

to the

and Sundqvist,32 who demonstrated in vivo that bacte-

OOOOE4 Dornelles-Morgental et al. Month 2011

ria remaining after biomechanical preparation can re-colonize the root canals.

NaOCl completely eradicated E. faecalis immedi-ately after instrumentation but allowed bacterial growth7 days later, as previously observed by other au-thors.30,31,33 The NaOCl � citric acid combinationpresented lower antibacterial activity than pure NaOClin both periods. A possible explanation for this wouldbe the release of chlorine gas when the 2 substanceswere mixed, before irrigating the root canal, as sug-gested by Baumgartner and Ibay.34

The NaOCl � apple cider vinegar combination, after7 days, presented higher CFU/mL counts than NaOClalone. Thus, association of NaOCl with acidic sub-stances did not prove advantageous, contrasting withthe report by Camps et al.12 It should be pointed out,however, that those authors used a higher concentrationof NaOCl (10%) in association with chloridric acid, toobtain a 2.5% NaOCl solution with neutral pH.

Plain apple cider vinegar promoted a significant re-duction in the numbers of E. faecalis following instru-mentation, but the final bacterial counts were similar tothe initial values.

The efficacy of 2% chlorhexidine against E. faecalishas been previously demonstrated, both in vitro and invivo.33,35 Our results demonstrate similar antibacterialactivity for 2% chlorhexidine and 2.5% NaOCl, con-trasting with the findings by Dametto et al.,31 whoreported absence of bacteria 7 days after preparationand irrigation with chlorhexidine. However, these au-thors allowed an incubation period of only 7 days afterinoculation, which may have prevented greater pene-tration of E. faecalis in the root canal system.

Peracetic acid solutions in concentrations rangingfrom 0.2% to 10.0% have been tested.19,24-26 Lottanti etal.24 detected, by scanning electron microscopy (SEM),dentin erosion after the use of 2.25% peracetic acid. Forthis reason, a lower concentration (1%) was chosen forthis study. Its activity against E. faecalis was similar tothat of NaOCl and chlorhexidine in both the postinstru-mentation and final periods.

None of irrigating solutions tested was able to per-manently eradicate E. faecalis from the root canal sys-tem. Further research focusing on tissue-dissolvingproperties, dentin wall cleaning ability, and biocompat-ibility are also important when evaluating potentialirrigants for use in endodontics.

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tanomaru@uol.com.br