International Endodontic Journal [1997} 30, 91-95

Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation
E. K. SIREN. M, P. P, HAAPASALO^ K, RANTA, P. SALMI & E, N, J, KEROSUO
Department ofCariology. Institute of Dentistry, University of Helsinki. Helsinki. Finland and "Division ofEndodonties. BentalPaculty, University of Oslo. Oslo. Norway

Summary The relationship between bacteriological findings and clinical treatment procedures was investigated in root canal treatment cases that were selected for bacteriological investigation by general dental practitioners iin Finland. The cultures were sent to the Oral Microbiological Service Laboratory at the Institute of Dentistry in Helsinki. Two groups of teeth were selected based on the type of infection present in the root canal system. The 'enteric bacteria' group consisted of 40 sequential cases wbere Enterococcus faecalis and/or other facultative enteric bacteria or VseuAomonas sp. were found in the samples in pure culture (35%) or together with other types of bacteria. The group 'nonenteric bacteria' consisted of 40 sequential cases where only non-enteric bacteria were found. The dentists who had sent the bacteriological! samples received a questionnaire where they were asked about the treatinent protocol and procedures. A total of 70 out of 80 questionnaires were returned. If the root canals had been unsealed at some point during the treatment, enteric bacteria were found morefrequentlythan in canals with an adequate seal between the appointments. Of cases with enteric bacteria 55% had been open during the treatment, while in the group where only non-enteric bacteria were found 30% had been open. Enteric bacteria were also morefrequentlyisolated in cases with a high number of appointments before sampling. In the enteric bacteria group 35% ofthe samples were taken at the 10th visit or later, while the corresponding percentage in the non-enteric group was 3%. In addition, the number of retreatment cases was significantly higher. 12 out of 34. in the enteric bacteria group than in non-enteric bacteria group, which was five out of 36.
Correspondeoce: Markus Haapasalo, Division of Endodontics, Dental Faculty, P,0, Box 1109, Blindern, N-0317 Oslo, Norway,
1997 Blackweil Science Ltd

Other clinical parameters showed no differences between the two groups. The results emphasize the importance of controlled asepsis throughout the root canal treatment. Keywords: drainage, Enterobacter, Enterobacteriaceae infections, Enterococcus faecalis, root canal therapy.

Introduction
Non-surgical root canal treatment of apical periodontitis consists of chemomechanical mstnimentation of the canal with removal of necrotic pulp tissue, disinfection of the canal and final sealing with a bacteria-tight material. Occasionally the treatment cannot be successfully completed and the root canal infection persists in spite of the therapy. The most common reasons for failures in conservative root cana! therapy are related to problems in instrumentation, however, occasionall}' bacteria resistant to conservative therapy of good quality may also be involved. Numerous studies have shown that the bacterial flora in root canal infections is polymicrobial w^ith a strong dominance of anaerobic bacteria (Sundqvist 1976, Haapasalo 1986a, Sundqvist et al. 1989), However, root canal treatment changes the ecological conditions in the root canal, for example the redox potential and nutritional factors. Information about persistent endodontic infections is limited (Bender & Selzer 1952,, Grahnen & Krasse 1963,, Engstrom 1964, Goldman & Pearson 1969, Haapasalo et al. 1983, Ranta et al. 1988),, Persistent endodontic infections are often caused by Enterococcus faecalis (Bender & Selzer 1952. Grahnen & Krasse 1963, EngstrSm 1964, Goldman & Pearson 1969), but also Gram negative enteric rods such as Enterobacter sp,. and KlebsieUa sp,. Pseudomonas or Gram positive facultatives such as Actinomyces spp, may be 91

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E. K. Siren et al. other facultative enteric bacteria or Pseudomonas sp. were found from the samples alone or together with other types of bacteria. The second group consisted of teeth where only non-enteric bacteria were found. Initially, 40 cases were chosen for both groups in the order in which the samples arrived at the Oral Microbiological Service Laboratory. Bacteriological procedures General practitioners were asked to follow the written instructions of the Oral Microbiological Service Laboratory. The disinfection of the sampling area was recommended to be carried out according to the procedures described earlier (MoUer 1966) and the samples taken with sterile forceps using sterile paper points inserted into the canal at the apical portion of the root canal. After sampling the paper points were immediately placed into the transport medium, VMGA ni gel (MoUer 1966), The transport medium contained glass beads with a diameter of 3 mm to facilitate mixing and homogenization of the sample prior to cultivation. Al! samples were cultivated within 3 days of sampling. The transport media were thoroughly shaken in a mixer (Vortex, Scientific Industries Inc., Springfield, MA. USA) and serially diluted to 10~^ and 10"* in sterile peptone water. One aliquot of 0.3 mL of undiluted medium and several aliquots of 0.1 mL ofthe undiluted and the two serial dilutions were plated onto several media using sterile plastic spreaders. Aliquots were distributed on Brucella agar plates (BEL Microbiology Systems, Cockeysville. MD. USA) enriched with 5% defibrinated horse blood, 5 mgL~^ of haemin and 10 mgL"-* of vitamin K,, TSBV agar plates (Tryptic-soy-agar enriched with 10% horse serum, 75 mgL"-' of bacitracin and 5 tngL"^ ofvancomycin; Slots 1982) and chocolate agar plates (Tryptic-soy-agar base with 10% defibrinated horse blood; Haapasalo 1986b), and incubated at 37C in 5% COj and anaerobically (Haapasalo 1986b), Bacterial colony types were enumerated, isolated and identified. Identification of pure cultures was done according to established procedures as described previously (Haapasalo et al 1983, 1986). Facultative streptococci were characterized by haemolytic patterns and growth in bile esculin medium (Difco laboratories, Detroit, MI, USA). API 20 E and API 20 NE (Biomerieaux sa, Marcy/Etoile, France) were used in the identification of facultative Gram negative enteric rods and pseudomonas, and API 20 Strep tests (Biomerieaux sa) were used for the identification of some streptococcus isolates.
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involved (Haapasalo ctal, 1983, Happotien et al 1985, Tronstad et al. 1987, Ranta et al 1988). These bacteria may be encountered either as monoinfections or together with one or a few other species. Enterococcus faecalis has been reported to sustain an alkaline environmeat (Bystrom et al, 1985, Haapasalo & 0rstavik 1987, 0rstavik & Haapasalo 1990) and to sun'ive long periods without nutrients. A previous study has shown that Enterococcus faecalis was the most resistant to calcium hydroxide of the species tested (Bystrom et al, 1985). Information about the susceptibilities of other enteric bacteria to local root canal medicaments is limited (Ranta e(l. 1988,0rstavik& Haapasalo 1990). The differences in the compositions of the root canal flora in cases responding well to endodontic therapy and in persistent cases of apical periodontitis may be due to: 1 a small amount of enteric bacteria is already present in the infected canal at the beginning ofthe therapy and their relative proportion increases during the treatment as other bacteria are more susceptible to therapy or 2 enteric bacteria enter the root canal during the treatment because of (i) inadequate isolation of the working area, (ii) a leaking temporary filling or (iii) the root canal has been left open for drainage. It has been recently suggested that the frequency of isolation of enteric bacteria is higher in endodontic retreatment cases compared to teeth treated for the first time (Molander et al. 1994). However, there are no earlier studies about the differences in the preceding treatment procedures, such as choice of materials and medicaments for example, between cases of persistent root canal infections with either enteric or non-enteric bacteria. The aim of the present investigation was to study the relationship between clinical treatment procedures and the occurrence of facultative enteric bacteria in root canal infections.

Material and methods

Selection of cases The relationship between the occurrence of specific bacterial floras and the use of different clinical treatment procedures was studied in root canal treatment cases selected for bacteriological investigation by the treating dentist. Microbial samples were sent to the Oral Microbiological Service Laboratory at the Institute of Dentistry in Helsinki. Two groups were selected based on the type of infection present in the root canal. The first group consisted of teeth where Enterocoecus faecalis and/or

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Collection of clinical information A questionnaire was sent to the dentists about the treatment protocol and procedures: endodontic diagnosis before the treatment; occurrence and type of symptoms during the treatment; use and type of systemic and local medication; use and type of temporary seal between the appointments including possible incidents of unsealed canals; the number of dentists who had given treatment to the tooth; and the number of appointments before the bacteriological sample. Statistical analysis Differences in treatment procedures between the two groups were statistically analysed by a chi square test.

Table 2 Other bacteria found with enteric bacteria in mixed infections. Total number of mixed infections was 22 Facultative Gram positive hactBria Streptococcus sp, Actinomyces odontolyticus Micrococcm sp. Staphylocoirus sp. Facultative Gram negative hacteria Neisseria sp, Anaerobic Gram positive bacteria Feptostreplococcus micros Anaerobic Gram negative bacteria Fusobacterium nucleatum PremteEa intermedia/nigrescens Campylobacter rectus Yeasts Candida sp, 12 2 1 1 2 2 7 4 1 5

Table 3 Bacteria and yeasts fouiid in the non-enteric group

Results
Enterococcus faecalis was the most common finding in the enteric bacteria group. In 33% of the cases where E. faecalis was isolated, it appeared as a monoinfection (Table 1), Other facultative enteric bacteria found were
Enterohacter cloacae, Enterobacter sakazakii, Enterobacter agglomerans, KlebsieUa oxytoga, Acinetobacter sp, and

Pseudomonas aeruginosa, of which four out of 10 were monoinfections (Table 1), Other species most often found together with enteric bacteria were Streptococcus sp,,
Fusobacterium nucleatum and Prevotella intermedia/

nigrescens (Table 2), These bacteria were the most common findings also in the non-enteric group (Table 3), A total of 70 out of 80 questionnaires were returned (88%), The number of replies in the study groups 'enteric bacteria' and 'non-enteric bacteria' were 34/40 and 36/40, respectively. Enteric bacteria were more frequently isolated in cases with a high number of visits before sampling and the difference was statistically significant (P < 0,05), In the cases with 10 or more visits enteric bacteria were found in 12 out of 13 samples (Table 4), If the root canals had been unsealed at some time during the treatment, enteric bacteria were found
Table 1 The frequencies of isolations and occurrence as monoinfections of facultative enteric bacteria. Total number of cases was 34 Species Enterococcus faecalis Enlerobacter cloacae Enterobacier agglomerans Enterobacter sakazakii KlebsieUa oxytoga Acinetobacter sp. Pseudomonas aeruginosa Total 24 5 1 1 1 1 1 Monoinfections 8 2 1 0 0 0 1

Facultative Gram positive bacteria Streptococcus sp. Actinomyces sp. Stiiphylococcus sp. Micrococcus sp. Lactobacillus sp. Facultative Gram negative bacteria Neissiria sp. Anaerobic Gram positive bacteria Peptostreptococcus micros Anaerobic Gram negative bacteria Fusobacterium nucleatum Prevotella intermedia/nigrescens Porphyramonas gingivalis Yeasts, Candida sp.

26 9 S 1 1 6 5 10 4 1 4

Table 4 Number of visits before the bacteriological sample was taken Number of visits 0-4 5-9 10 or more Total Enteric bacteria Non-enteric bacteria n 17 IS 1 36 Total

/ IS 12 34

24 33 13 70

^2 = 12.,843 : P < 0 . 0 5 .

more frequently than in canals with an adequate seal between the appointments (Table 5), The proportion of retreatment cases ui the enteric bacteria group was 12 out of 34, while in the non-enteric bacteria group only five of 36 cases were retreatments (Table 6), No statistically significant differences between the groups were detected in other clinical parameters i,e, pre-treatment status, symptoms, local and systemic medications and type ofthe temporary Ming (P > 0,05),

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E, K. Siren et al. infection in a necrotic root canai by pure cultures of bacteria usually fails (Fabricius et al. 1982), It is interesting that in the present material 35% of the infections caused by enteric bacteria were monoinfections. In the non-enteric group most species found were similar to those described in necrotic pulps before the treatment (Sundqvist 1976, Haapasalo 1986a), Enteric bacteria were chosen as the study bacteria because they are considered to be difficult to eliminate from infected root canals. Enterococci are often implicated in persistent root canal infections {Bender & Selzer 1952, Engstrom 1964, Haapasalo et al 1983), Calcium hydroxide is the most commonly used root canal dressing in Scandinavia, Also, in cases studied here it was used in 65 of 70 treatments. In an earlier study, of the 27 strains tested the most resistant species to calcium hydroxide was Enterococctis faecalis (Bystrom et al. 1985), The killing rate was dependent on the pH, cells survived at pH 11.5 for over 6 min, but not at pH 12,5, Although E. faecalis is seldom found in primary infections, it is the species most often isolated in retreatment cases of apical periodontitis (Molander et al 1994). Also, in the present study more retreatm.ent cases were found in the enteric bacteria group. The information available about the susceptibility of the other enteric bacteria to calcium hydroxide is limited and based on case reports (Ranta et al. 1988). In vitro studies have indicated that other medicaments, such as chlorhexidine and camphorated paramonochlorophenol may be more effective in the elimination of E, faecalis from the root canal sj'stem than calcium hydroxide (Bystrom 1986, Haapasalo & 0rstavik 1987, Heling et al. 1989, 0rstavik & Haapasalo 1990), Very little is known about the susceptibility of other enteric bacteria or Pseudomonas sp, to various local medicaments. This kind of information is needed for the more effective treatment of persistent endodontic infections caused by these bacteria. In the present study significantly more retreatment cases were found in the enteric bacteria group. This supports the findings of a previous study showing a higher proportion of enterococci in retreatment cases (Molander et al. 1994), However, in our study the majority of cases in the enteric bacteria group were treated for the first time. The reasons for the occurrence of enteric bacteria in persistent root canal infections is poorly understood. The observations of the present study that both a high number of visits and lack of an adequate seal significantly increased the probability of finding enteric bacteria in the root canal, indicate that these bacteria enter the root canal during the treatment. When the number of appointe 1997 Blackweii Science Ltd. InternanoniJBndoiionacJourmil, 30.91-95

Table 5 Bacterial iindings in root canal samples according to the adequacy of the seal of the temporary filling betiveen the visits. Five cases were excluded due to the unknown state of the seal Enteric bacteria n Sealed canal Unsealed canal*' Total 14 17 31 Non-enteric bactieria n 24 10 34

X" = 4.317: F < 0.05. "The canal had been open between the visits at least once before the bacteriological investigation. TaHe 6 Distribution of retreatment cases in the enteric bacteria and the non-enteric bacteria groups Enteric bacteria Retreatment Primary treatment Total X- = 4 . 3 5 7 : P < 0 . 0 5 . 12 22 34 Non-enteric bacteria 5 31 36

Discussion
Iaforoiation available about enteric bacteria in endodontic infections is scarce. However, it seems that they are relatively rare. This is probably the main reason why comprehensive studies of root canal infections caused by enteric bacteria has not been published. The collection of the present material was possible hecause endodontic samples from all of Finland are diagnosed at the Oral Microbiological Service Laboratory of the University of Helsinki, The reliability of information collected retrospectively by a questionnaire must be regarded with caution. However, the fact that the dentists were using microbiological investigation as a part of endodontic treatment and that the percentage of returned questionnaires was high may indicate that the dentists were dedicated to good quality endodontic treatment and documentation. The rubber dam is an integral part of root canal treatment in maintaining a high level of asepsis. Use of the rubber dam isi taught in universities, hut unfortunately in clinical practice the use of the rubber dam is often neglected. In the present study the usage was not enquired about for two major reasons: (i) the number of returned questiomiaires might have been considerably lower and (ii) the reliability of answers to this question might have been in sokie doubt. Previous studies on uncomplicated apical periodontitis have clearly indicated that the infections are caused by a minimum of two different species, monoinfections being rare (Sundqvist 1976, Fabricius et al. 1982, Haapasalo 1986a,b), Furthermore, experimental studies made in animals have also indicated that establishing an

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ments before sampling in the present study was 10 or more the difference between the two groups was strikiog as only one of the 13 cases belonged to the non-enteric group. This is in accordance with an earlier study where Enterococcus faecalis was not found in the initial sample of a case of apical periodontitis but was discovered in the root canal after 2 weeks of camphorated paramonochlorophenol treatment (Bystrdm 1986). In theory, the high number of appointments could also be a sequel to the presence of enteric bacteria in the root canal and not vice versa. Although we believe this is not the case, the present study cannot rule out either possibility. Our results also support the widely accepted clinical practice that in problematic endodontic treatments, specific new diagnosis and strategic changes in treatment plan should be employed much earlier than at the 10th appointment. In nearly half of the cases with enteric bacteria the fillings were reported to have been present during the whole treatment. Preparing a bacteria-tight temporary filling requires great clinical care. Clinical experience has shown that temporary fillings may leak for various reasons. Therefore, it is impossible to lcnow exactly the qualit>' ofthe seal in cases where no missing temporary fillings were reported. In many countries it has been a general clinical regimen for years that in the case of acute apical abscess the root canal should be left open for drainage. Moreover, many endodontic textbooks suggest that teeth with symptomatic apical periodontitis should not be sealed in an acute situation, but should remain open for rapid and reliable relief of pain. However, clinical observations suggest that exudation is seldom so persistent that open drainage is needed and that the best emergency treatm.ent is complete chemomechanical instrumentation ofthe root canal (Tronstad 1991. ESE 1994). In conclusion, our results support the view that root canals should not be left open during endodontic therapy and emphasize the importance of maintaining a high level of asepsis throughout the treatment.

BvsTEOM A (1986) Evaluation ofendodontic treatment of teeth with apical periodontitis, Umea, Sweden: Umea University. (PhD Thesis).
BYSTROM A. CLAESSONR, SDNDQVIST G (1985) The antihacterial effect

of camphorated paramonochlorophenol, camphorated phenol and calcinm hydroxide in the treatment of infected root canals. Endodontics andDental Traumatology 1, 170-5. ENGSTROM B (1964) The significance of enterococd in root canal treatment. OdontologiskRevy IS, 87-106,
EDROPEAN SOCIETY OF ENDODONTOLOGY (1994) Consensus report of

the European Society of Endodontologj' on quality guidelines for endodontic treatment. International Endodontic Journal 2 7 . 1 1 5 - 2 4 .
FAERICIUS L, DAHLEN G, HOLM SE, MOLLER AfR (1982). Influence of

combinations of oral bacteria on periapical tissues of monkeys. Scandinavianjournal of Dental Research 90', 20f>-6. GOLDMAN M, PEAKSOK AH (1969) Postdebridement bacterial flora and antibiotic sensitivity. Oral Surgery, Oral Medicine and Oral Pathology 28,897-905. GRAHNEN H , KRASSE B (1963) The effect of instrumentation and flushing of non-vital teeth in endodontic therapy, Odontologisk Rcvy 14,167-77, HAAPASALO M . (1986a) The genus Bacteroides in human dental root canal infections: taxonomic, ultrastructural, and clinical studies, Helsinki. Finland: University of Helsinki. (PhD Thesis). HAAPASALO M (1986b) Baeteroides buccae and related tasa in necrotic root canal infections, Jouinal of Clinical Microbiology 24, 940-4, HAAPASALO M, 0RSTAVIK D (1987) In vitro infection and disinfection of dentinal tubules. Journal of Dental Research 66,13 75-9,
HAAPASALOM. RANTA K. RANTA H (1983) Facultative Gram-negative

enteric rods in persistent periapical infections, Acta Odontologica .Seandinanca 91. 458-63.
HAAPASALO M. RANTA H , RANTA K, SHAH H (1986) Black pigmented

Bacteroides spp, in human apical periodontitis, Jnfectinn and Immunity 5 3 , 1 4 9 - 5 3 .

HAPPONEN R P . SODERLING E, VlANDER M, LINKO-BCETTHNEN L

FELLINIEMI LJ (1985) Immunocytochemical demonstration of Actinomyces species and Arachnia propionica in periapical infections. Journal ofOral Pathology 14, 405-13.
HELINGI, SOMMEEM, STEINB,ERGD. SELAMN, FRIEDMAKM (1989)In

vitro evaluation of chlorhexidine efficacy as an intracanal medication (abstract)./ournoJo/JiniiodOTiUes 15, 176,

MOLANDER A. REIT C, DAHLEN G. KVIST T (1994) Microbiological

ex,ainination of root filled teeth with apical periodontitis (Abstract). International Endodontic Journal 2, 104, MOLLER AJR (1966) Microbiological examination of root canals and periapical tissues of human teeth. Odontologisk Tidskrift 74, 1-380. 0RSTAVIK D. HAAPASALO M (1990) Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endodontics andDental Traunmtology 6.142-9.
RANTA K. HAAPASALO M, RANTA H (1988) Monoinfection of root

Acknowledgements
We thank Dr odont. Harald Eriksen for his helpful criticism and suggestions during the preparation of this manuscript. This study was supported financially by NorFA.

References
BENDER NA, SELZER S (1952) Combination of antihiotics and fungicides used in treatment of the infected pnlpless tooth. Journal of the American Dental Association 45. 29 3-300.
1997 Biaclcwell Science Ltd, InternnaiinaJEndDiJoiiUc/ournoi, 3 0 , 9 1 - 9 5

canal with Pseudomonas aeruginosa. Endodontics and Dental Traumatology 4, 269-72. SLOTS J (1982) Selective medium for isolation of Actinobacillus actinomycetenicomitans. Journal of Clinical Periodontology 15, 60b-9. SuNDQViST G (1976) BacterioiojicaJ studies of necrotic pulps. Omea, Sweden; Umea University. (PhD Thesis). SDNDQvrsT G, JOHAN.SSON E, SjOGREN U (1989) Prevalence of biackpigmented Bacteroides species in root canal infections. Journal of Endodontics 15,13-9. TRONSTAD L (1991) Clinical Endodontics, 1st edn, Stuttgart, Germany: Georg Thieme Verlag,
TROKSTAD L. BARNETT F . Riso K, SLOTS J (1987) Extraradicular

endodontic infections. Endodontics and Dental Traumatology i,

86-90.