Microorganisms from Canals of Root-Filled Teeth with Periapical Lesions

Microorganisms from canals of root-filled teeth with periapical lesions

E.T.Pinheiro,B.P.F.A.Gomes,C.C.R.Ferraz,E.L.R.Sousa,F.B.Teixeira& F. J. Souza-Filho Endodontic Department, Piracicaba Dental School, State University of Campinas, UNICAMP,Piracicaba, SP,Brazil

Abstract frequently recovered bacterial species. Obligate anaerobes accounted for 42.6% of the species and the most Pinheiro ET, Gomes BPFA, Ferraz CCR, Sousa ELR, frequently isolated genera was Peptostreptococcus, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of which was associated with clinical symptoms root-filled teeth with periapical lesions. International Endodontic Jour- (P < 0.01). Signi¢cant associations were also observed nal, 36, 1^11, 2003. between: (a) pain or history of pain and polymicrobial Aim The objective of the present study was to identify infections or anaerobes (P < 0.05); (b) tenderness to the microbial £ora within root canals of teeth with percussion and Prevotella intermedia/P. nigrescens failed root-canal treatment and to determine the asso- (P < 0.05); (c) sinus and Streptococcus spp. (P < 0.001) ciation of the various species with clinical features. or Actinomyces spp. (P < 0.01); (d) coronally unsealed Methodology Sixty root-¢lled teeth with persisting teeth and Streptococcus spp. or Candida spp. (both with periapical lesions were selected for this study. During P < 0.01). nonsurgical endodontic re-treatment, the root-¢lling Conclusion The microbial £ora in canals after fail- material was removed and the canals were sampled. ure of root-canal treatment were limited to a small Microbial sampling, isolation and species determina- number of predominantly Gram-positive microbial tion were performed using advanced microbiological species. Facultative anaerobes, especially E. faecalis, techniques for anaerobic species. The association of were the most commonly isolated microorganisms, microbiological ¢ndings with clinical features was however, polymicrobial infections and obligate anae- investigated. robes were frequently found in canals of symptomatic Results Microorganisms were recovered from 51 root-¢lled teeth. teeth. In most cases, one or two strains per canal were Keywords: endodontic failure, microbiology, root- found. Of the microbial species isolated, 57.4% were canal therapy. facultative anaerobic species and 83.3% Gram-positive microorganisms. Enterococcus faecalis was the most Received 4 June 2001;accepted16August 2002

procedures have not met a satisfactory standard for con- Introduction trol and elimination of infection (Nair et al. 1990, 1999, Bacteriaortheirproductsareconsideredtobetheprimary Lin et al.1991). Persisting bacteria in root canals may be aetiological agents of pulpal necrosis and periapical those originally present inthe necrotic pulps that survive lesions (Kakehashi et al.1965,Mo« lleret al.1981,Takahashi the biomechanical procedures, which may be located in 1998). Therefore, their elimination is one of the most missed canals or uninstrumented areas of the canals important steps in root-canal treatment. In most cases (Fukushima et al. 1990, Ida & Gutmann 1995, Sjo« gren failure of root-canal treatment occurs when treatment et al. 1997). Conversely, bacteria may originate from the oral cavity,contaminate the root canal during treatment owing to inadequate aseptic control (Siren et al.1997),or Correspondence: Dr Brenda P.F. A. Gomes, PhD, MSc, BDS, Endodontia, invadetheroot-¢llingviacoronalleakageafterroot-canal Faculdade de Odontologia de Piracicaba, FOP-UNICAMP,Avenida Lime- ira 901, Piracicaba 13414 - 018, SP, Braz il (Tel.: þ55 19 3412 5215, fa x: treatment (Torabinejad et al.1990,Maguraet al. 1991, þ55193412 5218; e-mail: [email protected]). Ray & Trop e 1995, C heu ng 1996).

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36,1^11,2003 1 Microorganisms from canals of root-filled teeth Pinheiro et al.

Although it has been reported that nonmicrobial fac- nonsurgical endodontic re-treatment. A detailed medi- tors may be implicated in root-canal treatment failure, cal and dental history was obtained from each patient. the literature suggests that persistent intraradicular or Patients who had received antibiotic treatment during secondary infections are the major causes of the failure the last 3 months orhad ageneral disease were excluded of root-canal treatment (Siqueira 2001). Recent studies from the study. The Ethical Committee in Research of using advanced microbiological techniques for anaero- the Dental School of Piracicaba approved a protocol bic species (Molander et al. 1998, Sundqvist et al. 1998, describing the specimencollection for this investigation, Peciuliene et al. 2000, 2001) have revealed the composi- and all patients signed informed consent to participate tion of root-canal microbiota after failed treatment dif- in the study. fers from that normally found in untreated teeth. The Sixty teethwere included; all had beenpreviously root necrotic pulp presents a polymicrobial £ora character- ¢lledand showed radiographic evidence ofapicalperiod- ized by a wide variety of combinations of bacteria, aver- ontitis. Failure of root-canal treatment was determined aging 4^7 species per canal, predominantly anaerobic, onthe basis of clinicaland radiographical examinations. with approximately equal proportions of Gram-negative Most of the teeth (90%) had been root-canal-treated and-positive bacteria (Sundqvist etal.1989,Baumgartner more than 4 years ago; in six cases teeth had been root & Falker1991, Sundqvist1992a,b, Sato et al.1993, Gomes ¢lled more than 2 years ago and the patients presented et al. 1994, 1996a,b; Gomes 1995, Baumgartner et al. with persistent symptoms and/or discomfort to percus- 1999). In contrast, the microbial £ora detected in sion. The following features were recorded for each previously root-¢lled teeth with apical periodontitis can patient, so that they could be correlated with the micro- be characterized as monoinfection with predominant- bial ¢ndings: tooth type, clinical symptoms, presence ly Gram-positive microorganisms, with approximately or absence of a sound coronal restoration (i.e. a perma- equal proportions of facultative and obligate anaerobes nent restoration that clinically and radiographically (Sundqvist et al.1998). Molander et al. (1998) found that appeared sealed), caries, sinus, swelling of periodontal facultative anaerobic species predominated amongst the tissues, tenderness to percussion, mobility, periodontal isolates (69% of identi¢ed strains). Enterococcus faecalis status of the tooth, status of the root canal in terms of werethemostcommonlyisolatedspeciesfromrootcanals whether dry or wet (the term ‘wet canal’ in this study of teeth with failed endodontic treatment (Molander et al. means presence of exudate), and the radiographic qual- 1998,Sundqvist etal.1998,Peciuliene etal.2000,2001). ity of the root-canal ¢lling. Coronal restorations were Whereas follow-up studies on the root-canal treat- categorized as defective if there were open margins, frac- ment report success rates of 85^96% (Swartz et al.1983, ture or recurrent decay. Initial root ¢llings were classi- Sjo« gren et al. 1990, Smith et al. 1993), the literature has ¢ed as good if no voids were present and were within indicated that the success of re-treatment of teeth with 2 mm of the radiographic apex. If one or more of these apical periodontitis is lower, with an overall success rate criteria were not met, these were classi¢ed as poor of 66% (Allen et al. 1989, Hepworth & Friedman 1997). (Ray & Trope1995). Molander et al. (1998) suggested that this poorer prognosis in root-canal re-treatments may be associatedwith Sampling procedure di⁄culties inthe eliminationof the particularmicro£ora in cases of root-canal treatment failure. A thorough All coronal restorations, posts and carious defects were knowledge of this micro£ora could guide new strategies removed. After access cavity preparation, the teeth were to combat infection, leading to a better prognosis for individually isolated from the oral cavity with a rubber root-canal re-treatments. The aim of the present study dam, and disinfected with 5.25% sodium hypochlorite. was to investigate the microbial £ora of teeth with failed The solution was inactivated with 5% sodium thiosul- root-canal treatment and the association of constituent phate in order to avoid interference with bacteriological species with clinical features. sampling. Aseptic techniques were used throughout root-canal treatment and sample acquisition. In each case, a single root canal was sampled in order to con¢ne Materials and methods the microbial evaluation to a single ecological environ- ment. In multirooted teeth, the root with the periapical Clinical material lesion was selected. If there were periapical lesions in Patients were selected from those who attended the all roots, the wider canal was selected. The root ¢ll- Piracicaba Dental School, SP, Brazil, with a need for ing was removed using Gates^Glidden drills (Dentsply

2 International Endodontic Journal, 36,1^11,2003 ß 2003 Blackwell Publishing Ltd Pinheiro et al. Microorganisms from canals of root-filled teeth

Maillefer, Ballaigues, Switzerland) and endodontic ¢les Columbia agar plates and incubated aerobically at without the use of chemical solvents. Irrigation with 37 8Cfor2days. sterile saline solution was performed in order to remove After incubation, eachplatewas examinedandthe dif- any remaining materials and to moisten the canal prior ferent colony types subcultured onto plates to obtain to sample collection. pure culture. The colonial appearance was used for For microbial sampling, a sterilepaper point wasintro- selecting the colonies for further study. Pure cultures duced into the full length of the canal (as determined were then initially identi¢ed according to their Gram with a preoperative radiograph), and kept in place for morphology, ability to produce catalase and gaseous 60 s. Inthe cases that had been previously irrigated with requirements. saline, as many paper points as possible were used to Gaseous requirements were determined as follows: absorb all liquid or £uid inside the canal. The canal ori- each colony obtained by anaerobic incubation was used ¢ce was £ushed with nitrogen gas during sampling. to inoculate two plates of 5% sheep blood Columbia agar. The paper point samples from the root canal were trans- One was incubated for 2 days aerobically and the other ferred immediately to a transport medium ^ VMGA III for the same length of time anaerobically.The respective (Mo« ller1966, Dahle¤ n et al.1993) and transported within plates were then compared. Strict anaerobes were those 15 min to an anaerobic workstation (DonWhitley Scien- whichareable togrowonly under strictlyanaerobic con- ti¢c, Bradford, UK) in the microbiology laboratory. The ditions. average time between sample collection and laboratory These procedures permit the primary identi¢cation of processing was 4 h. the strain as Gram-positive or -negative, coccus or bacil- lus, catalase-positive or-negative, and aerobic and anaerobic. Based on these primary results, the appropriate Microbial isolation kit for identi¢cation was selected. Inside the anaerobic workstation, the transport media, containing glass beads with a diameter of 3 mm to facil- Microbial species determination itate mixing and homogenization of the sample, were shaken thoroughly in a mixer for 60 s (Agitador MA The following identi¢cation kits were used for primary 162-M A RC ON I, Sa‹ o Paulo, SP,Brazil). Serial10-fold dilu- speciation of individual isolates: Rapid ID 32 A (Bio Mer- tions were made up to1/104 in fastidious anaerobe broth ieux, Marcy-l’Etoile, France) for obligate anaerobic (FAB, Laboratory M, Bury, UK) and 50 mL of each serial Gram-negative and -positive rods; RapID ANA II System dilution were plated onto several media using sterile (Innovative Diagnostic Systems Inc., Atlanta, GA, USA) plastic spreaders. for obligate anaerobic Gram-positive cocci; API Staph Obligate anaerobes and facultative anaerobes were (Bio Merieux, Marcy-l’Etoile, France) for staphylococci cultured nonselectively on plates containing 5% de¢bri- and micrococci (Gram-positive cocci, catalase-positive); nated sheep blood^fastidious anaerobe agar (FAA, Rapid ID 32 Strep (Bio Merieux, Marcy-l’Etoile, France) Laboratory M, Bury, UK) incubated at 37 8Cinanatmo- for streptococci (Gram-positive cocci, catalase-nega- sphere of 10% H2,10%CO2 and 80% N2 for 2, 5 and tive); Rapid NH System (Innovative Diagnostic Systems 14 days. Selecting for Gram-positive anaerobes and acti- Inc., Atlanta, GA, USA) for Eikenella, Haemophilus, Neis- nomycetes involved the use of a 5% de¢brinated sheep seria and Actinobacillus. blood-FAA þ nalidixic acid (NAL, 0.001% w/v, Labora- These kits are based on distinct biochemical tests tory M, Bury, UK) agar plate at 37 8C anaerobically, for (enzymatic reactions). Each kit, therefore comprises 2,5 and14 days. Selecting for Gram-negative anaerobes rows of individual cupules (each containing a di¡erent involved the use of a 5% de¢brinated sheep blood^ substrate) to which inoculum is added. After a period FA A þ NAL þ vancomycin (VAN, 0.00025% w/v, (4 h for most species but18 h for Staphylococcus spp.) of Laboratory M, Bury, UK) agar plate at 37 8C anaerobi- incubation, and subsequent addition of the reagents, cally, for 2, 5 and 14 days. Selection for clostridia and precipitation or colour changes appear in individual other anaerobes involved the use of a 5% blood- cupules. Typically, a positive test is observed as a colour FA A þ neomycin (NEO,0.0075% w/v neomycin, Labora- change when a chromogenic substrate in an individual tory M, Bury, UK) agar plate at 37 8C anaerobically, for cupule is hydrolysed and liberates a coloured product. 2,5 and14 days. The results were read visually and identi¢cation of To detect aerobes and facultative anaerobes the sam- the strains was interpreted with the Analytical Pro¢le ples were inoculated onto 5% de¢brinated sheep blood- Index.

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36,1^11,2003 3 Microorganisms from canals of root-filled teeth Pinheiro et al.

For the black-pigmenting Gram-negative anaerobes, Table 1 Microorganisms found in 60 canals after removalof the following additional tests were also performed: (a) the root ¢lling £uorescence under long-wave (366 nm) UV light; (b) Microbial No. of root Percentage of haemagglutination of 3% sheep erythrocytes; (c) lactose species canals root canals fermentation ^ by application of the £uorogenic subs- E. faecalis 27 45 trate 4-methylumbelliferyl-b-galactoside (SigmaChemi- E. faecium 11.7 cal Co., St Louis, MO-M-1633), according to Alcoforado S. constellatus 58.3 et al. (1987); and (d) trypsin-like activity by application S. sanguis 46.7 S. mitis 23.3 of the synthetic £uorogenic peptide 7-(N-carbobenoxy- S. anginosus 23.3 glycylglycylarginin-7-amido)-4-methylcoumarinhydro- S. mutans 11.7 chloride (C-9396) (Nakamura et al.1984, Slots1987). S. oralis 11.7 S. salivarius 23.3 P. p r e v o t i i 610.0 Statistical analysis P. m i c r o s 58.3 P. m a g n u s 11.7 The data collected for each case (clinical and radio- P. saccharolyticus 11.7 graphic features, and the bacteria isolated) were entered A. naeslundii 46.7 intoa spreadsheet (QUATTROPro,BorlandInternational A. odontolyticus 35 Inc., Scotts Valley, CA, USA) and analysed statistically A. viscosus 35 Prevotella buccae 35 using SPSS for Windows (SPSS Inc., Chicago, USA). The P. intermedia/nigrescens 35 Pearson chi-square test or the one-sided Fisher’s exact P. melaninogenica 11.7 test, as appropriate, was chosen to test the null hypoth- P. corporis 23.3 esis that there was no relationship between: (a) clinical P. loescheii 11.7 symptoms(painorhistoryofpain),clinicalsigns(sinus, Propioni. acnes 46.7 P. propionicum 11.7 swelling, canal exudate, tenderness to percussion), pre- Gemella morbillorum 46.7 sence and quality of coronal restorations, quality of the Veillonella spp. 4 6.7 previous endodontic treatment; and (b) the presence of Fusobacterium necrophorum 11.7 any particular species of bacteria in previously root- F.nucleatum 11.7 treated canals. Fusobacterium spp. 1 1.7 Lactobacillus acidophilus 23.3 S. lentus 23.3 Results Haemophilus aphrophilus 11.7 Eubacterium lentum 11.7 Forty-seventeethwere single-rootedand13multirooted. Bifidobacterium spp. 1 1.7 Five patients presented for treatment with acute pain. Clostridium subterminale 11.7 Lactococcuslactiscremoris 11.7 The remainder had no spontaneous pain, although 20 Capnocytophaga spp. 1 1.7 gave a historyof previous pain.Tenderness to percussion Candida spp. 2 3.3 was present in 20 teeth and a sinus tract was detected in ¢ve patients. Thirty-seven teeth were restored with a permanent coronal restoration, 22 of which were defec- two species and 15 (25%) were polymicrobial infections tive and15 sound. Ten teeth were restored with tempor- consistingofthreeormorespeciespercanal.Theclinical ary restorative materials showing breakdown or and radiographic features and microbial ¢ndings of each fracture, and 13 had no restorations. Previous gutta- case are presented in Table 2. percha ¢llings were present in all teeth. Upon radio- Facultative anaerobes accounted for 57.4% of all graphic examination, 22 teeth had good root ¢llings, species isolated, and obligate anaerobes accounted for whilst 38 had poorly obturated canals. 42.6%.There was a predominance of Gram-positive spe- Onehundredand eight cultivable isolates belonging to cies (83.3%). The prevalence of bacterial genera found 37 di¡erent species (Table 1) were recovered from the inthe60rootcanalsisshowninFig.1.E. faecalis was 60 root canals examined after root ¢lling removal. Nine the bacterial species most frequently recovered, being (15%) root canals had no cultivable bacteria; 28 found in 27 (52.94%) of the 51canals with bacteria, 18 (46.7%) cases presented a single microorganism, in 18 times in pure culture. of them Enterococcus faecalis was the only microorgan- Clinical features werepresent inteethassociated with ism isolated from the canal; 8 (13.3%) cases presented 60 canals as follows: spontaneous pain (5/60), history

4 International Endodontic Journal, 36,1^11,2003 ß 2003 Blackwell Publishing Ltd ß

03BakelPublishing Ltd Blackwell 2003 Table 2 Clinical and radiographic features and bacterial ¢ndings

Case no. T TAT R P HP TTP Sw PR W/D Si RCF Bacteria

122>4NRNNNNYDNPES. sanguis 241>4TRNNNNYDNPES. sanguis 3 3212TRNNNNYDNGE^ 4 1220PRNNNNYDNPES. salivarius 522>2TRNNNNYWYPES. mitis, E. faecalis, A. naeslundii 6 158GRYYYNYWNPEP. m i c r o s , P. p r e v o t i i 714>4TRNNNNYDNGE^ 811>4NRYYYNYDNGEP. saccharolyticus 946>4GRNNNNYWNPEPropioni. acnes, E. faecalis 10 2 2 >4TRNNNNYDNPE^ 11455PRNNNNYDNPE^ 12 11 >4GRNNNNYDNPEP. buccae, P. micros, L. acidophilus, E. faecalis 13 2 1 9 NR Y Y Y N Y W N PE P.intermedia,P.melaninogenica,P.corporis,F.necrophorum,P.prevotii,P.magnus,S. constellatus, E. faecalis, S. lentus 14 4 5 >4NRY Y Y N Y W N GEVeillonella ssp., S. mutans, L. acidophilus, Candida spp. 153115PRNNNNYDNGE^ 16 2 2 >2GRNNYNYDNPES. mitis, S. anginosus, G. morbillorum 17445GRYYYYYDNGE^ 181310PRNYYNYDNPEE. faecalis 19 4 6 >2NRN Y Y N Y W N PE P.buccae,P.loescheii,A.naeslundii 20125PRNNNNYDNPEE. faecalis 211220PRNYNNYDYPEFusobacterium ssp., S. oralis, A. viscosus, G. morbillorum

22 12 >2TRNNYNYDNPEVeillonella ssp., P. prevotii, A. odontolyticus, G. morbillorum, S. lentus, H. aphrophilus al. et Pinheiro 233720PRNNYNYDNGEE. faecalis 24325GRNNNNYDNPEP. p r e v o t i i 25225PRNYYNYDNGEE. faecalis 261210PRNYYNYDYPEP.prevotii,E.faecalis,S.sanguis nentoa nootcJournal, Endodontic International 272110PRNNNNYDNPEE. faecalis root-filled teeth of canals Microorganisms from 28 15 >4PRNNNNYDNPES. constellatus 291110PRNNNNYDNPEE. faecalis 302210PRNNNNYDNGE^ 31 21 5 NR N Y Y N Y D Y GE S. constellatus 32467NRNNYNYDNPEPropioni. acnes, S. constellatus, Streptococcus ssp. 33 46 >4TRYYYNYDYPEP. intermedia, P. micros, Bifidobacterium ssp., A. naeslundii, Streptococcus ssp. 34457TRNYNNYDNGEP. buccae, P. micros, P. prevotii, F. nucleatum, E. faecalis 35375PRNYYNYDNGEE. faecalis

36 36118PRNNNNYDNGEVeillonella ssp., A. odontolyticus, A. viscosus, G. morbillorum ,1^11,2003 37 35 >4GRNNYNYDNPEA. viscosus 38226NRNNNNYDNPEPropioni propionicum,Veillonella ssp., A. naeslundii, E. faecalis, Candida spp. 39118NRNYNNYDNPE E. faecalis 5 6 irognssfo aaso root-filled teeth of canals Microorganisms from nentoa nootcJournal, Endodontic International

Table 2 continued

36 Case no. T TAT R P HP TTP Sw PR W/D Si RCF Bacteria ,1^11,2003 403330PRNNNNYDNGE^ 412112TRNNNNYDNPEE. faecalis 42235PRNNNNYDNPEE. faecalis 43455NRNYNNYDNGES. anginosus, Capnocytophaga spp. al. et Pinheiro 443230PRNNNNYDNGEE. faecalis 454230PRNNNNYDNGEE. faecalis 46374PRNYYNYDNGEP. intermedia, E. faecalis 47215TRNYNNYDNGEE. faecalis, Lact. lactis cremoris 483412PRNNNNYDNPEE. faecalis 49225GRNNNNYDNPEE. faecalis 501215GRNYYNYDNPEE. faecalis 512111GRNNNNYDNPEE. faecalis 521111GRNNNNYDNPEE. faecalis 531411NRNNNNYDNPEA. odontolyticus, S. salivarius 54 46 >4GRNNNNYDNPEP. corporis, P. micros, Propioni. acnes 55 47 >4GRNNNNYDNPEE. lentum, S. sanguis 56243NRNNYNYDNGEE. faecalis 57126GRNNNNYDNGEPropioni. acnes 58455NRNNNNYDNGES. constellatus, E. faecium 59418PRNNNNYDNPEC. subterminale 60 46 >2GRNYYNYDNPE^

Abbreviations used:T,tooth;TAT, time after treatment (years);R, restoration; GR, good restoration; PR, poor restoration;TR, temporary restoration; NR, no restoration; P,pain; HP,history of pain;TTP,tenderness to percussion; Sw, swelling; PR, periapical radiolucency;W, wet canal; D, dry canal; Si, sinus tract; RCF, root-canal filling; GE, good endodontic filling; PE, poor endodontic filling;Y, yes; and N, no. ß 03BakelPublishing Ltd Blackwell 2003 Pinheiro et al. Microorganisms from canals of root-filled teeth

Figure 1 Prevalence of bacterial genera found in 60 teeth with failed endodontic treatment.

of pain (20/60), tenderness to percussion (20/60), sinus Discussion (5/60), swelling (1/60) and wet canal (5/60). Root-¢lled teeth with pain or history of pain were associated with In the present study, microorganisms were recovered the presence of polymicrobial infections and anaerobes from 51of the 60 teeth examined. The ¢nding that 15% (P < 0.05), including Prevotella spp. (especially P. i n t e r- of root canals had no cultivable bacteriawas not entirely media/P. nigrescens)andFusobaterium spp. (P < 0.05). unexpected; earlier studies were unable to isolate bac- Spontaneous pain was especially associated with the teria from 55.6% (Sundqvist et al.1998),26.6% (Molander isolation of Peptostreptococcus spp. (P < 0.01). Ten- et al.1998),20% (Peciuliene et al.2000) and17.5% (Peciu- derness to percussion was observed in 20 root canals liene et al.2001) of teeth where chloroform was not used and was associated with P. intermedia/P. nigrescens to dissolve the gutta-percha. However, failure to detect (P < 0.05). Five cases of sinus were associated with bacteria does not prove theirabsence. Althoughthe sam- the ¢ndings of Streptococcus spp. (P < 0.001) and pling techniques and laboratory procedures used in this Actinomyces spp. (P < 0.01), especially A. naeslundii study have been previously shown to be highly e¡ective (P < 0.001). Coronally unsealed teeth showed a signi¢- (Gomes et al. 1994, 1996a,b,c; Gomes 1995), it is possible cant association with Streptococcus spp. (P < 0.01) and that some microorganisms could have been lost, espe- Candida spp. (P < 0.01). cially if the number of microorganisms present in the There were no signi¢cant relationships between the rootcanalwas verylowor if they werepresent ininacces- radiographic status of the endodontic treatment and sible areas suchas anatomical branches and apical areas the presence of any particular species of bacteria obliterated by the previous treatment. Furthermore, (P > 0.05). However, poorly obturated canals were some microorganisms could have been removed toget- found to be associated with polymicrobial infections her with the previous root ¢lling and debris (Sundqvist (P < 0.05). et al. 1998).

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36,1^11,2003 7 Microorganisms from canals of root-filled teeth Pinheiro et al.

The results showed that amongst the 51 teeth with are more resistant to instrumentation and to antiseptic positive culture, 28 cases presented a single species, 8 agents, and therefore can be expected to persist morefre- cases presented two species, and15cases presentedpoly- quently inthe rootcanalafter inadequate root-canalpre- microbial infections consisting of 3 or more species per paration and obturation (Cavalleri et al. 1989, Gomes canal. These ¢ndings agree with those reported by et al.1996c, Molander et al.1998). Persisting microorgan- Sundqvist et al. (1998) who found monoinfections in 19 isms or their products can maintain an infectious pro- of 24 teeth with bacteria. The same authors suggested cess and cause treatment failure. According to that teethwith poor root-canal treatment are morelikely Molander et al. (1998), facultative anaerobes can survive to have a £ora similar to that found in untreated canals in a quiescent phase with low metabolic activity for a than the teeth with apparently well-cleaned canals. period of time, and factors such as coronal leakage can The present investigation involved teeth with good and change the nutritional conditions and contribute to bac- poor root-canal ¢llings, and showed a signi¢cant asso- terial growth. ciation between poorlyobturated canals and polymicro- E. faecalis was the bacterial species most frequently bial infections (P < 0.05). It also involved symptomatic recovered, being found in 52.94% of canals with bacter- or asymptomatic teeth, and again polymicrobial infec- ial growth. This ¢nding agrees with those reported by tions were associated with spontaneous pain or history Molander et al. (1998) and Sundqvist et al. (1998) who, of pain (P < 0.05). respectively,found E. faecalis in 47 and 38% of previously Of the total bacterial species isolated, 57.4% were root-treated canals with positive culture. Peciuliene facultative anaerobes and 83.3% Gram-positive species. et al. (2000, 2001) reported a higher isolation frequency This ¢nding is in accordance with earlier studies by of this microorganism:70 and 64%, respectively. Sundqvist et al. (1998) and Molander et al. (1998), who In the present study, E. faecalis was isolated in pure found 58 and 69% facultative anaerobes and 87 and culture in18 of the 27 cases where this species was pre- 74.3% Gram-positive microorganisms, respectively. In sent. Peciuliene et al. (2000, 2001), respectively, isolated the present study,the facultative anaerobic genera most thismicroorganisminpureculturein5of14cases frequently isolated were Enterococcus, Streptococcus and in11of 21teeth. Studies have shown the pathogeni- and Actinomyces, in agreement with previous ¢ndings city of this species in monoculture (Fabricius et al. (Sundqvist et al. 1998). 1982). E. faecalis has demonstrated the capacity to sur- Obligate anaerobes accounted for 42.6% of the species vive inanenvironment inwhichtherearescantavailable and the most frequently isolated genera was Peptostrep- nutrients and in which commensality with other bac- tococcus, which was signi¢cant associated with clinical teria is minimal (Sundqvist et al.1998). It has been postu- symptoms. Prevotella spp. (especially P. i n t e r m e d i a/ lated that a virulence factor of E. faecalis in failed P. nigrescens)andFusobacterium spp. were frequently endodontically treated teeth may be related to the ability isolated from canals of root ¢lled teeth with pain or his- of E. faecalis cells to maintain the capability to invade tory of pain. P. intermedia/P. nigrescens was associated dentinal tubules and adhere to collagen in the presence with tenderness to percussion. Several studies investi- of human serum (Love 2001). gating the microbiology of primary infected root canals E. faecalis makes up a small percentage of the £ora in also reported a relationship between the presence of the original infection and may be favoured by the chan- anaerobes with clinical symptoms, whilst oral strepto- ged ecology in the root canal and establish infections cocci and enteric bacteria were frequently isolated from which are di⁄cult to treat (Engstro« m 1964, Goldman & asymptomatic teeth (Yoshida et al. 1987, Baumgartner Pearson 1969, Cavalleri et al. 1989, Sundqvist 1992a). 1991, Gome s et al. 1994, 1996a). Gomes et al. (1996c) showed that E. faecalis was more fre- The presence of a sinus tract was associated with quently recovered from the canals in later appointments either Streptococcus spp. or Actinomyces spp., especially after biomechanical treatment procedures. Sundqvist A. naeslundii. In all but one case the root canals had a et al. (1998) reported that from nine cases in which E. fae- mixed £ora. Anaerobes belonging to Prevotella, Peptos- calis was isolated in initial samples during root-canal treptococcus and Fusobacterium species and also faculta- re-treatment, ¢ve root canals contained this bacterial tive anaerobes such as E. faecalis were also isolated. species after cleaning and shaping and the use of cal- Gomes et al. (1994,1996a) also found that the microbiota cium hydroxide as medicament. Molander et al.(1998) present inteethwith a sinus was predominantly mixed. suggested that the use of calcium hydroxide as an intra- Facultative anaerobic and Gram-positive bacteria, canal dressing may be one of the reasons for selection such as Enterococcus, Streptococcus and Actinomyces, of E. faecalis in root canals, as these bacteria are able to

8 International Endodontic Journal, 36,1^11,2003 ß 2003 Blackwell Publishing Ltd Pinheiro et al. Microorganisms from canals of root-filled teeth survive high pH values (Bystro« m et al.1985, Haapasalo & Conclusion Orstavik 1987), and could have a negative impact on the prognosis. However, Peciuliene et al. (2000) found a In conclusion, the £ora of canals with failure of root- high frequency of E. faecalis in teeth in which calcium canal treatment comprised a limited numberofpredomi- hydroxide was not used for treatment, indicating that, nantly Gram-positive microbial species. Facultative rather than previous chemical treatment, it is the ecolo- anaerobes, especially E. faecalis,werethemostcom- gical condition of the incompletely ¢lled root canal that monly isolated microorganisms from teeth. However, is important for the presence of this microorganism. polymicrobial infections and obligate anaerobes were According to Siren et al. (1997), Enterococcus spp. and frequently found in canals of symptomatic root ¢lled other enteric bacteria may enter the root canal during teeth. This information should direct further studies at treatment owing to inadequate isolation of the working methods to eliminate infection during re-treatment, in area, leakage of the temporary ¢lling or in cases where order to improve the prognosis. the root canal had been incorrectly left open for drai- The present analysis is exploratory.It must be stressed nage. The same study showed that the number of failed that owing to a large number of statistical tests, it is pos- cases was signi¢cantly higher in teeth that harboured sible that some associations are statistical artefacts. Par- enteric bacteria than in teeth that harboured nonen- ticular relationships that have been identi¢ed will need teric-bacteria. to be corroborated with a con¢rmatory study. Such a As the root-canal treatment of the teeth investigated studyenables the constructionof a more speci¢chypoth- in this study were carried out by unknown operators, esis and this, inturn, facilitates the employment of amul- microbial samples were not collected before root-canal tivariable method to eliminate possible confounding ¢lling. Therefore, it is not possible to determine whether factors. the microorganisms recovered had persisted after prior root-canal treatment, had contaminated the root canal Acknowledgements during treatment or had invaded the root ¢lling via coronal leakage after root-canal treatment. This work was supported by the Brazilian agencies In the present study,coronal leakage (bydefective cor- FAPESP (1996/5584-31999/05641-52000/13686-8, 2000/ onal restorations, old temporary restorative materials 13689-7) and CNPq (520277/99-6). or coronally unsealed teeth) was detected in most of the teeth (45/60), and may have in£uenced the microbial References ¢ndings. In the cases of absence of a coronal restoration, signi¢cant relationships were found with Streptococcus Alcoforado GAP, McKay TL, Slots J (1987) Rapid method for spp. (P < 0.01) and Candida spp. (P < 0.01). detection of lactose fermentation oral microorganisms. Oral Although fungi have usually not been found in the Microbiology and Immunology 2,35^8. Allen RK, Newton CW,Brown CE (1989) A statistical analysis of initial £ora of root-canal infections, their presence is surgical and nonsurgical endodontic retreatment cases. Jour- more common in persistent infections after root-canal nal of Endodontics15, 261^6. preparation, probably as a result of contamination dur- Baumgartner JC (1991) Microbiologic and pathologic aspects of ing treatment, or in cases of root-¢lled teeth with ther- endodontics. Current Opinion in Dentistry1,737^43. apy-resistant periapical lesions (Nair et al.1990).Inthe BaumgartnerJC, FalkerWA(1991)Bacteria inthe apical 5 mmof present investigation, Candida spp. was recovered in 2 infected root canals. Journal of Endodontics17,380^3. of 60 cases. This agrees with those of Sundqvist et al. Baumgartner JC,Watkins BJ, Bae KS, XiaT(1999) Association of (1998) and Molander et al. (1998) who reported the pre- black-pigmented bacteria with endodontic infection. Journal sence of this microorganism in 2 of 54 teeth and in 3 of Endodontics 25,413^5. of 100 teeth examined, respectively. Peciuliene et al. Bystro« m A, Claesson R, Sundqvist G (1985) The antibacterial (2001) found Candida albicans in 6 of 40 teeth studied. e¡ect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of Waltimo et al. (1999) have shown that Candida spp. are infected root canals. Endodontics Dental Traumatology 1, highly resistant to antiseptics and disinfectants com- 170 ^ 5. monly used as endodontic irrigants and medicaments. Cavalleri G, Cuzzolin L, Urbani G, Benoni G (1989) Root canal These microorganisms have been isolated in pure cul- micro£ora: qualitative changes after endodontic instrumen- tures from root-canal infections, a fact that may indicate tation. Journal of Chemotherapy 1,101^2. that these are pathogenic in apical periodontitis (Wal- Cheung GSP (1996) Endodontic failures ^ changing the timo et al.1997). approach. International DentalJournal 46,131^8.

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