Basic Research—Biology

The Starvation Resistance and Biofilm Formationof Enterococcus faecalis in Coexistence withCandida albicans, Streptococcusgordonii, Actinomyces viscosus,or Lactobacillus acidophilus

Yan Gao, MS,*

†Xiaoqiong Jiang, MS,*

†Dongjia Lin, MS,* Yanhuo Chen, MS,*

and Zhongchun Tong, PhD*†

Abstract

Significance

The multispecies community is conducive to theresistance to starvation of Enterococcus faecalisand biofilm formation in root canals, which mightbe one of the primary reasons E. faecalis isfrequently found in root-filled teethwith apical peri-odontitis.

Introduction: Enterococcus faecalis is the mostfrequently detected species in root canal–treated teeth,and it is able to survive under starvation conditions.However, persistent periapical disease is often causedby multispecies. The aim of this study was to explorethe survival of E. faecalis in starvation conditionsand biofilm formation with the 4 common pathogenicspecies. Methods: A dual-species model of Candidaalbicans, Streptococcus gordonii, Actino-myces viscosus, or Lactobacillus acidophilusin combination with E. faecalis was established andallowed to grow in phosphate-buffered saline for theexamination of starvation survival. Cefuroxime sodiumand vancomycin at a concentration of 100 mg/L wereadded into brain-heart infusion plate agar to countthe 2 bacteria separately in the dual species. Scanningelectron microscopy was used to observe the dual spe-cies and multiple species on the root canal dentin ofbovine teeth for 48 hours. A confocal laser scanningmicroscope was used to show the 4 groups of dual-species biofilms on substrates with glass bottoms for48 hours. Results: E. faecalis was more resistant tostarvation in coexistence with C. albicans, S. gor-donii, A. viscosus, or L. acidophilus, and S. gor-donii was completely inhibited in coexistence with E.faecalis. The dual-species biofilm showed that E.faecalis formed thicker and denser biofilms on theroot canal dentin and glass slides in coexistence withS. gordonii and A. viscosus than C. albicansand L. acidophilus. Conclusions: The multispeciescommunity is conducive to the resistance to starvationof E. faecalis and biofilm formation in root canals. (JEndod 2016;42:1233–1238)

From the *Department of Operative Dentistry and EndodonticsSun Yat-sen University, Guangzhou, Guangdong, China.

Address requests for reprints to Dr Zhongchun Tong, DepartmentGuangzhou 510055, Guangdong, China. E-mail address: t_z_c19770099-2399/$ - see front matter

Copyright ª 2016 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2016.05.002

JOE — Volume 42, Number 8, August 2016

Key WordsBiofilm, dual species, Enterococcus faecalis, starvation

Pathogenic bacteria area major cause of end-

odontic infections andperiradicular periodonti-tis. Microorganisms inthe root canal are involvedin dynamic processes, andbacterial species dependon available nutrition, oxy-gen levels, and local pH in

the root canal system (1). In a primary root canal infection, the open root canal pro-vides an available resource for microbes through direct communication with the oralcavity. Anaerobic gram-negative bacteria and several gram-positive rods are often foundin the root canal (2). However, in root-filled canals with persistent periapical disease,an obvious decrease in the availability of resources in the root canal occurs becausechemomechanical preparation and intracanal medication are introduced and theroot canals are sealed. Gram-negative bacteria gradually decrease, and gram-positivebacteria are more frequently present, including streptococci, Parvimonasmicra, Acti-nomyces species, lactobacilli, Propionibacterium species, and Enterococci (3–7).

A few studies have indicated that Enterococcus faecalis is the most frequentlydetected species in root canal–treated teeth, and it promotes persistent disease(8–11). E. faecalis has the ability to adapt to harsh environmental changes, such asan extreme alkaline pH, salt concentrations, deprivation of nutrition, antimicrobialresistance, and growth in the root canal as a biofilm (12, 13). The properties ofE. faecalis determine its survival in root-filled canals. In the past, the frequent occur-rence of monocultures of E. faecalis has raised suspicion that this bacteriummay be thesole organism that persists in root canals, but monoinfections seldom occur in nature.Chronic periapical disease is often caused by multiple species, and the interactions ofthese species are very complex (14, 15). Interspecies interactions in multispeciesbacterial biofilms exhibit social behavior and generate the protective effect of

, Guanghua School of Stomatology and †Guangdong Provincial Key Laboratory of Stomatology,

of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University,@163.com

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Basic Research—Biology

biofilms (16). E. faecalis frequently inhabits this undernourished envi-ronment of root-filled canals and depends not only on its own proper-ties but also the synergistic and antagonistic interactions betweenspecies in the biofilm. At present, the interspecies interactions of multi-species biofilms in root-filled canals are still poorly understood, andfurther information on interspecies interactions is needed.

The root-filled canal is an environment with deficient nutrition,and microbes are grown in biofilms in root canals. Lactobacillus, Acti-nomyces, Streptococcus, and Candida have been reported to be path-ogenic species that are isolated in retreated root canals (2, 17, 18).In this study, dual-species models of E. faecalis and Lactobacillusacidophilus, Actinomyces viscosus, Streptococcus gordonii, orCandida albicans were used to evaluate their resistance to starvation,and the biofilm structure was observed with scanning electron micro-scopy and a confocal laser scanning microscope.

Materials and MethodsBacterial Culture

E. faecalis ATCC 29212, C. albicans ATCC 90028, L. acidophilusATCC 4356, A. viscosus ATCC 15987, and S. gordonii ATCC 10558 wereused in this study. The bacteria were streaked from a frozen stock cul-ture onto their respective plates at 37�C for 48 hours. E. faecalis, A.viscosus, and S. gordonii were grown on brain-heart infusion agar(BHI; Difco Laboratories, Detroit, MI) plates; C. albicans were grownon yeast peptone dextrose agar (Difco) plates; and L. acidophilus weregrown on de Man, Rogosa, and Sharpe agar (Difco) plates. A single col-ony of bacteria was inoculated into 5 mL of the corresponding liquidbroth and cultured at 37�C under anaerobic conditions up to the expo-nential phase.

Specimen PreparationBovine incisors were used to measure the biofilm formation of

dual species on root canal dentin. Fresh bovine incisors were ex-tracted from animals that were slaughtered for commercial purposesin a slaughterhouse in China and stored in saline. Our study exertedno influence on the fate of the animals at any point. The specimenpreparations were similar to those described in a previous study(19). Briefly, the crown and root apex of the teeth were removed,and the length of the remaining tooth root was 10 mm. Access prep-arations were instrumented to a size 60 accompanied by irrigationwith 5.25% sodium hypochlorite and 17% EDTA to remove thepulp tissue and the smear layer. The roots were longitudinally splitinto 2 parts, and the root canal dentin was exposed to bacteria to allowbiofilm formation. The prepared specimens were sterilized in an auto-clave at 121�C for 20 minutes before use as a substrate in the dual-species coculture.

Antibacterial AssayAccording to the different bacterial resistance to antibiotics,

100 mg/L cefuroxime sodium was added to BHI agar to distinguishE. faecalis and A. viscosus, E. faecalis and L. acidophilus, or E. fae-calis and S. gordonii, and 100 mg/L vancomycin was added to BHI agarto distinguish E. faecalis and C. albicans in dual-species coculture. Theminimum inhibitory concentration (MIC) was determined as describedin a previous study (20). In short, 10 mL bacterial culture, 140 mL BHIbroth, and 50 mL 2-fold diluted antibiotics were added to 96-wellmicroplates and cultured at 37�C under anaerobic conditions for24 hours. TheMIC is defined as the lowest concentration of an antibioticthat inhibits bacterial growth with an increase in optical density <0.050under standard conditions. TheMIC was determined at least in triplicateon a different day.

1234 Gao et al.

The Starvation Assay of Dual SpeciesIn this assay, E. faecalis and C. albicans (EC), E. faecalis and S.

gordonii (ES), E. faecalis and A. viscosus (EA), and E. faecalis and L.acidophilus (EL) established 4 dual-species models. In a mixture ofdual species, the final concentrations of both bacteria were adjustedto 108 colony-forming units (CFUs)/mL according to their respectivegrowth curves. The bacterial culture of the dual species was centrifugedat 5000 rpm at 4�C for 5 minutes. The supernatant was discarded. Thecell deposit was washed twice with PBS, and then resuspended in PBSfor the starvation assay of dual species. The total bacterial amount of thedual species was obtained on 1, 2, 4, 7, 14, and 28 days of starvation bybacterial plate counts. At each time point of starvation, 100mL dual spe-cies was 10-fold diluted and spread on BHI agar for 48 hours of incu-bation. The total bacterial amount of dual species was counted.Meanwhile, to separately count E. faecalis in the ES, EA, and EL groups,100 mL dual species was 10-fold diluted and spread on BHI agar con-taining 100 mg/L cefuroxime sodium only to allow E. faecalis colonyformation. To count C. albicans separately in the EC group, 100 mLdual species was 10-fold diluted and spread on BHI agar containing100 mg/L vancomycin only to allow C. albicans colony formation.The bacterial count of the other species in the dual-species modelwas calculated by subtracting the bacterial quantity in BHI agar withantibiotics from the total bacterial quantity. All determinations wereperformed at least 3 times on different days.

Root Canal Infection with Dual SpeciesRoot canal infections with dual-species biofilm were performed

under the condition of abundant nutrition because starvation affectsbacterial growth and does not form a typical biofilm structure. TheEC, ES, EA, and EL groups and a group of mixture of the 5 bacteria(E. faecalis, C. albicans, S. gordonii, A. viscosus, and L. acidophilus)were inoculated into 12-well plates (Corning Costar, Corning, NY) con-taining 3 mL BHI broth, respectively, and the final concentration of eachbacteria was 105 CFU/mL. Five root specimens were transferred to eachwell with inoculated culture medium and incubated at 37�C for48 hours. The specimens were rinsed twice with sterile PBS to removeplanktonic and loosely adherent bacteria and to ensure that only theattached biofilm remained. The specimens were prepared for scanningelectron microscopy (SEM [E-1010; Hitachi, Ibaraki, Japan]) by fixa-tion in 2.5% glutaraldehyde and dehydration in a series of acetonitrilesolutions (50%, 70%, 80%, and 90% for 20 minutes each and 100% for20 minutes twice). The specimens were dried and coated with gold, anda randomly selected area of biofilm was observed on the root canaldentin using SEM.

Confocal Laser Scanning MicroscopyThe bacterial competition in dual-species biofilms was observed

under the condition of abundant nutrition using a confocal laser scan-ning microscope (CLSM). Petri dishes with glass bottoms (diameter =35 mm [Hangzhou Shengyou Biotechnology, Zhejiang, China) wereconsidered to be a substrate to allow for biofilm formation. The EC,ES, EA, and EL groups of dual species at a 1:1 bacterial ratio were addedinto the Petri dishes with 2 mL tryptic soy broth (Difco) supplementedwith 1% glucose, and the final concentration of each bacteria was108 CFU/mL. After 48 hours of incubation at 37�C, the biofilm at the bot-tom of the well was gently washed twice with sterile PBS and stained witha mixture of 6 mmol/L SYTO 9 stain and 30 mmol/L propidium iodide(PI) at room temperature in the dark for 15 minutes according to thespecifications of the L13152 kit (LIVE/DEAD Bac-Light BacterialViability Kit; Molecular Probes, Eugene, OR). Images of the stainedspecimens were captured using a Carl Zeiss CLSM (Carl Zeiss

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TABLE 1. Minimum Inhibitory Concentration of 5 Test Bacteria against Vancomycin and Cefuroxime Sodium

Antibiotics (mg/L)E. faecalisOG1RF

C. albicansATCC90332

A. viscosusATCC15987

L. acidophilusATCC4356

S. gordoniiATCC10558

Vancomycin 8 >1024 2 >1024 1Cefuroxime sodium 512 >1024 <0.5 4 <0.5

Basic Research—Biology

MicroImaging Inc, Thornwood, NY) and ZEN software (ZEN 2012 LightEdition, Carl Zeiss MicroImaging, Inc). SYTO 9 and PI were excited at488 nm and 543 nm, respectively. The 3-dimensional (3D) bacterialbiofilm was scanned along the z-axis. All images are shown at 400�magnification. SYTO 9 stain generally labels all bacteria in a populationas those with intact membranes and those with damagedmembranes. Incontrast, PI penetrates only bacteria with damagedmembranes, causinga reduction in SYTO 9 stain fluorescence when both dyes are present.Therefore, with a mixture of the SYTO 9 and PI stains, viable bacteriawith intact cell membranes stain fluorescent green, whereas dead bac-teria with damaged membranes stain fluorescent red.

Statistical AnalysisStatistical analysis was performed using SPSS 18.0 software (SPSS

Inc, Chicago, IL). Raw bacterial colony counts were transformed tolog10 values to normalize the data. One-way analysis of variance andthe Tukey honestly significant difference test were used to comparethe survival rates of E. faecalis in the 4 groups of dual species and aloneat the time of starvation and the biofilm thickness of the 4 groups of dualspecies and E. faecalis alone for 48 hours. The Student t test was used tocompare the survival rates of C. albicans, S. gordonii, A. viscosus, andL. acidophilus in dual species and alone at the same time of starvation.P <.05 was considered statistically significant.

ResultsAntibacterial Assay

Vancomycin and cefuroxime sodium showed different antibacte-rial activities against E. faecalis, C. albicans, L. acidophilus, A. visco-sus, and S. gordonii (Table 1).

Dual-species Resistance to StarvationAt 1 and 2 days of starvation, no significant difference was

found between the survival rates of E. faecalis in dual species

Figure 1. The survival count of E. faecalis alone and in coexistence with C. albicanstarvation. The asterisks represent a statistically significant difference between the su(P < .05).

JOE — Volume 42, Number 8, August 2016

and alone (P > .05). E. faecalis generated greater resistance tostarvation in the presence of C. albicans, S. gordonii, A. viscosus,and L. acidophilus than alone after 4 days, and both showed sig-nificant differences (P < .05). The coexistence of C. albicanssignificantly improved the survival rates of E. faecalis in starvationconditions (Fig. 1).

In the starvation evaluation of dual species,C. albicans had a goodability to endure starvation whether in coexistence with E. faecalis oralone, and both showed no statistical difference at the same day of star-vation (P > .05) (Fig. 2A). E. faecalis completely inhibited S. gordoniiunder starvation conditions in dual species, and S. gordonii did not sur-vive for 1 day of starvation in the presence of E. faecalis. The survivalrates of S. gordonii showed a statistically significant difference in thepresence of E. faecalis or alone (P< .05) (Fig. 2B). In the dual speciesof E. faecalis and A. viscosus, the survival rates of A. viscosus showed arelative rise in comparison with them alone (Fig. 2C). Enduring starva-tion, the survival rate of L. acidophilus did not show a significant changein the presence of E. faecalis, but at 28 days of starvation, L. acidoph-ilus in coexistence with E. faecalis showed a lower survival rate thanalone (Fig. 2D).

Biofilm Morphology of Dual Species in Root CanalsThe biofilm structure of the 4 groups of dual species and a group

of multiple species in the root canal were examined by SEM. The cocul-ture of E. faecalis and C. albicans (Fig. 3A), E. faecalis and L. aci-dophilus (Fig. 3D), and a mixture of 5 strains (Fig. 3E) showed arelatively thin biofilm on the root canal dentin, and the dentinal tubuleswere still clearly recognized. Both E. faecalis and C. albicans wereobserved on the root canal dentin in coculture (Fig. 3A), whereas inthe coculture of E. faecalis and L. acidophilus, E. faecalis dominatedthe root canal dentin, and only a small quantity of L. acidophilus wasobserved (Fig. 3D). In the biofilm structure of the coculture of 5 strains,cocci, bacilli, and Candida appeared on the root canal dentin (Fig. 3E).E. faecalis and S. gordonii (Fig. 3B) and E. faecalis and A. viscosus

s, S. gordonii, A. viscosus, and L. acidophilus, respectively, for 1 to 28 days ofrvival count of E. faecalis in dual species and alone at the same day of starvation

Starvation Resistance of E. faecalis 1235

Figure 2. The survival count of (A) C. albicans, (B) S. gordonii, (C) A. viscosus, and (D) L. acidophilus, respectively, alone and in the presence of E. faecalis for1 to 28 days of starvation. The asterisks represent a statistically significant difference between the survival count of the tested bacterium in dual species and alone atthe same day of starvation (P < .05).

Basic Research—Biology

(Fig. 3C) formed a rather thick and dense biofilm, and a large numberof cocci covered the root canal dentin.

3D Biofilm Structure of Dual SpeciesA CLSM was used to scan the 3D biofilm structure of dual species

and detect alive and dead cells. The biofilms of dual species of

Figure 3. Morphology of the 4 groups of dual species and a multiple-species group(B) E. faecalis and S. gordonii; (C) E. faecalis and A. viscosus; (D) E. faecalisA. viscosus, and L. acidophilus. Images a, b, c, d, and e are local magnifications

1236 Gao et al.

E. faecalis and S. gordonii (Fig. 4B) and E. faecalis and A. viscosus(Fig. 4C) were significantly thicker than those of E. faecalis and C.albicans (Fig. 4A), E. faecalis and L. acidophilus (Fig. 4D), andE. faecalis alone (Fig. 4E), and there was a statistically significant dif-ference between biofilm thickness of ES or EA group and the controlgroup (Fig. 4F). (P< .05). In the group of E. faecalis and C. albicans(Fig. 4A) and E. faecalis and A. viscosus (Fig. 4C), some red dead

grown on the root canal dentin for 48 hours. (A) E. faecalis and C. albicans;and L. acidophilus; and (E) a group of E. faecalis, C. albicans, S. gordonii,of images A, B, C, D, and E, respectively.

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Figure 4. 3D scanning images of dual-species biofilms shown by confocal laser scanning microscopy. (A) E. faecalis and C. albicans, (B) E. faecalis and S.gordonii, (C) E. faecalis and A. viscosus, (D) E. faecalis and L. acidophilus, and (E) E. faecalis alone. Cells with intact membranes stain green, whereas cells withdamaged cell membranes stain red. All images are shown at 400� magnification using a Carl Zeiss CLSM and ZEN 2012 software. (F) Comparison of the biofilmthicknesses of the 4 groups of dual species and E. faecalis alone. *A statistically significant difference between biofilm thickness of the dual species and the control(P < .05).

Basic Research—Biology

cells were shown, and, in the other 3 groups, green viable cells domi-nated the biofilm.

DiscussionThe pathogenic bacteria of root-filled canals with apical periodon-

titis are frequently restricted to a few gram-positive bacteria, but persis-tent infection is often caused by polymicrobial action. AlthoughE. faecalis was frequently found in root-filled teeth with apical peri-odontitis, the strains were not isolated as a monoinfection in most cases(21, 22). Therefore, we explored the dual-species model of E. faecalisand the other 4 common species found in root-filled canals. To quantifyeither bacterium in dual species, 2 antibiotics, vancomycin and cefur-oxime sodium, at final concentrations of 100 mg/L were added to themedium to distinguish them according to their different MIC values.The species of MIC <100 mg/L did not grow, and the bacterial colonyon the plate agar was equal to the quantity of the other species.

The bacteria’s resistance to starvation may determine whether thebacteria survive in root-filled canals. The survival of a single bacteria,such as E. faecalis, Fusobacterium nucleatum, Peptostreptococcusanaerobius, Prevotella intermedia, or Pseudoramibacter alactolyti-cus and a fungus (C. albicans), has been previously examined(23–25). The starvation endurance of dual species was evaluated inour studies, and we found that E. faecalis showed more resistance tostarvation in the presence of C. albicans, S. gordonii, A. viscosus,and L. acidophilus, respectively, than alone. Under deficient nutrient

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conditions, the death and decomposition of C. albicans, S. gordonii,A. viscosus, or L. acidophilus might provide some essentialingredients for E. faecalis survival. Although the low nutrientcomposition does not meet the requirements of the growth ofE. faecalis, it may in part help the resistance to starvation ofE. faecalis. However, on the contrary, S. gordonii did not survive inthe presence of E. faecalis. Nutrient deficiency may activateinterspecies competition. E. faecalis is an Enterococcus spp, and itcan produce bacteriocins, such as enterocin and cytolysin, whichinhibit competitors in the microflora (26, 27). S. gordonii might besensitive to the bacteriocins produced by E. faecalis, and, thus, itwas inhibited in dual species. However, the reason that C. albicans,A. viscosus, and L. acidophilus have similar starvation kinetic curvesin the presence and absence of E. faecalis might be because of thethree microorganisms’ insensitivity to antibacterial agents producedby E. faecalis. This speculation needs to be examined further.

Multispecies biofilms of root canal bacteria have been described inprevious studies (28, 29). E. faecalis biofilms on the biotic and abioticsurface are usually unstable and easily disturbed. The dual-species bio-film model of root canals showed that E. faecalis formed a dense bio-film structure on the root canal dentin in the presence of S. gordoniiand A. viscosus, respectively. S. gordonii and E. faecalis in SEMwere not distinguished because of their similar morphologies, but wediscerned that E. faecalis dominated in the dual species of E. faecalisand A. viscosus, which was consistent with the results of Ch�avez de Pazet al (29), showing that E. faecalis OG1RF can inhibit Actinomyces

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Basic Research—Biology

naeslundii by using miniflow chambers and 16S ribosomal RNA filterin situ hybridization A. viscosus and A. naeslundii belong to the samespecies and may have similar properties. Although S. gordonii and A.viscosus may promote E. faecalis growth, we have not yet observeddense biofilm on the root canal dentin in a 5-species model. From anecological viewpoint, microorganisms maintain an ecological balancein microflora. E. faecalis may generate overproliferation in a dual-species model, but in a multispecies model, interaction between bacte-ria is very complex, and E. faecalis may keep a dynamic balancebecause of mutual competitive inhibition between bacteria. The afore-mentioned studies were performed with sufficient nutrition. However,abundant nutrition is not a normal state in root-filled canals. Under star-vation conditions, S. gordonii was completely inhibited by OG1RF, butA. viscosus showed better survival under starvation in coexistence withOG1RF than alone, in contrast to the abundant nutrition in which A.naeslundii or A. viscosus was inhibited (29). E. faecalis was moreresistant to starvation in coexistence with S. gordonii and A. viscosusthan alone. These interspecific interactions might be a reason why E.faecalis frequently dominates in root-filled canals.

Starvation and alkalinity are 2 important factors that influence theamount and species of microorganisms in the root canal system. Ourstudies indicated that E. faecalis was more resistant to starvation incoexistence with 4 other bacteria, and A. viscosus and S. gordonii pro-moted the survival of E. faecalis with abundant nutrition. The studies ofvan der Waal et al (30) showed that calcium hydroxide favors the pop-ulation of E. faecalis in dual-species biofilms in coexistence with Pseu-domonas aeruginosa. These studiesmay help explain the prevalence ofE. faecalis in persistent root canal infection; therefore, attention shouldbe paid to the root canal bacteria that facilitate the overproliferation ofE. faecalis and resistance to starvation with the microbiological factorsof persistent root canal infection.

AcknowledgmentsYan Gao and Xiaoqiong Jiang contributed equally to this work.We are grateful to Elsevier Language Editing Services for pro-

fessionally editing this article.Supported by grants from the Guangdong Natural Science

Foundation (no. 2014A030313026).The authors deny any conflicts of interest related to this study.

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