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University J Dent Scie 2016; No. 2, Vol. 1 ISOLATION OF ROOT CANAL PATHOGENS FROM PRIMARY ENDODONTIC INFECTION AND RETREATMENT CASES – A CLINICAL COMPARATIVE STUDY. 1 Vineet R.V., 2Moksha Nayak, 3Subbannayya Kotigadde, 4Beena Antony Senior Lecturer, Department of Conservative Dentistry & Endodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam, 2 Principal & Professor, Department of Conservative Dentistry & Endodontics, KVG Dental College & Hospital, Sullia 3 Professor, Department of Microbiology, K.V.G Medical College & Hospital, Sullia 4 Professor, Department of Microbiology , Father Muller Medical College, Mangalore 1 ABSTRACT Aim: The purpose of this study was to isolate and compare the prevalence of common root canal pathogens; Streptococcus mitis, Lactobacillus acidophilus and Enterococcus faecalis from primary endodontic infections and retreatment cases. Materials and method: 60 subjects scheduled for root canal treatment or retreatment were divided into two groups comprising of 30 subjects with primary endodontic infections and 30 subjects with failed endodontic treatment. Root canal samples were collected as per Moller's criteria using sterile paper point. Advanced microbiological culture techniques were used to isolate Streptococcus mitis, Enterococcus faecalis and Lactobacillus acidophilus from primary root canal infections and retreatment cases. Statistical analysis was done using Student's T test and Pearson's Chi-Square test. Results: A total of 50 cultivable isolates were recovered from 60 root canal samples. Among them 26 isolates were from primary infection and 24 isolates were from secondary infection. Streptococcus mitis(53.3%) was significantly more associated with primary infection. On the other hand Enterococcus faecalis (46.7%) was the most predominant microorganism in retreatment cases. Conclusion: Higher prevalence of Streptococcus mitis in primary infection and Enterococcus faecalis in secondary infection, suggest that specific type of root canal infection has an affinity to specific group of microorganisms. Knowledge regarding this association may be beneficial in adopting strategies to combat root canal infections for better prognosis. INTRODUCTION : Endodontic disease is the result of both the pathogenic effects of the microbes and the response of the host. The goal of clinicians is to disrupt and destroy the microbial ecosystem associated with the disease process. Understanding of microbial ecology of the pulp-dentine complex is important because it provides a rational basis for disease prevention and treatment.[1] Endodontic treatment involves treatment of both primary and secondary infections in the root canal system. Primary infections are caused by microorganisms that colonize the necrotic pulp tissue. It can be regarded as the initial or 'wild' infection and are the cause of primary apical periodontitis.[2] Clinical studies have reported a success rate of endodontic therapy ranging from 87% to 95%.[3] Secondary infections are caused by microorganisms that were not present in the primary infection, but were introduced in the root canal at some time after professional intervention[4]. University Journal of Dental Sciences Clinical Papers and Comparative Studies Keywords : Isolation, Streptococcus mitis, Lactobacillus acidophilus, Enterococcus faecalis, primary infection, retreatment cases. Source of support : Nil Conflict of interest: None The composition of the microflora of root canals differs in primary infection and retreatment cases of endodontic origin.[5] Microorganisms from dental caries have proved to be the most common causative factor of pulpal disease. Studies have shown that Streptococcus sp., Lactobacillus sp., Enterococcus sp. are strongly associated with dental caries.[6] A relatively wide range of bacteria have been isolated from root canals using standard culture techniques. However, only 50% of the bacteria in the oral cavity are cultivable.[7] Use of advanced anaerobic microbiological techniques and culturing in selective media have been proven to be beneficial in this regard. A selective medium contains antimicrobial agents that are capable of suppressing all other microorganisms other than one for which the medium is designed. Selective culturing constitutes an efficient technique to detect very low proportions of specific microorganism from a sample.[8] University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 06 University J Dent Scie 2016; No. 2, Vol. 1 To date there are no comparative data regarding the association of Streptococcus mitis, Lactobacillus acidophilus and Enterococcus faecalis in primary endodontic infection and in retreatment cases using selective media. In addition,the microbial flora that colonize the root canals vary with the geographic location of the patients studied.[9] Literature on microorganisms isolated from root canals in Indian population is sparse. Hence, it is prudent to study the gram positive microorganisms especially Streptococcus mitis, Lactobacillus acidophilus, Enterococcus faecalis from primary endodontic infections and retreatment cases using advanced culturing technique among Indian population. MATERIALS AND METHOD : Study population: Root canal samples were collected from 60 subjects who had been referred for root canal treatment or retreatment to the Department of Conservative dentistry and Endodontics, K.V.G. Dental College & Hospital, Sullia. The Ethical Committee of K.V.G Dental College & Hospital,Sullia approved the study protocol. The subjects were informed of the study protocol and written consent was obtained before the sampling procedure was performed. 60 subjects selected for the study were divided into two groups. Group 1: 30 subjects with primary endodontic infections. Group 2: 30 subjects with failed endodontic treatment requiring retreatment. cavity. It was then transferred to a vial containing 1ml of liquid transport medium and assessed for bacterial growth. If growth occurred the patient sample was disqualified from the study. All instruments used for access cavity preparation were sterile. In case of a treated canal, canal filling material was removed with the use of Gates Glidden drills and endodontic files without the use of any chemicals. Canal was rinsed with sterile saline to remove the remnants of filling material and debris, and to moisten the canal before sampling. Working length of the canal was determined radiographically using a 20 K-file 0.5mm short of the radiographic working length. Sampling was performed by placing a sterile paper point in the canal to its full length for 60seconds. In case of multirooted teeth, a single root canal was sampled in order to confine the microbial evaluation to a single ecologic environment. The criteria used to choose the canal for sampling were the presence of exudation, or in its absence, the largest canal or the canal associated with periapical radiolucency. Before sampling the selected canal of multirooted teeth, the entrance of the other canals was closed with sterile cotton pellets. Following removal from the canal, the paper point was immediately placed in a transport medium containing 3ml of sterile reduced transport fluid which was further transferred to Robertson cooked meat medium and glucose broth. Both males and females aged 20-70 years; immunocompetent subjects were included in the study. In group 1 patients with deep carious lesion involving pulp or with a diagnosis of pulpal necrosis were included. In group 2 patients requiring retreatment of endodontically treated teeth with a diagnosis of apical periodontitis, root filled teeth with radiographic evidence of periradicular disease and the termini of the root canal fillings atleast 2mm short of the radiographic apex were included in the study. Subjects with any systemic diseases, pregnancy and lactation, use of any antibiotics during past 3 months, immunocompromised patient, teeth that cannot be isolated with rubber dam, calcified canals and teeth having periodontal pockets greater than 4mm were excluded from the study. Root canal sampling: Aseptic techniques were used throughout the root canal sampling procedure as proposed by Moller. The tooth was isolated with rubber dam. All coronal restorations and carious lesions was completely removed. The tooth and surrounding field was rinsed using 30% hydrogen peroxide and then with 2.5% sodium hypochlorite solution for 30seconds. The solution was inactivated with sterile 5% sodium thiosulfate. To assess the efficacy of the disinfection procedure,a sterile cotton pellet was used to swab the access Figure 1: Root canal sampling technique University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 07 University J Dent Scie 2016; No. 2, Vol. 1 Bacterial culture method: The root canal samples obtained were then immediately submitted to the Department of microbiology, K.V.G Medical College and Hospital, Sullia for culturing. The root canal samples were shaken in a vortex mixer for 60 seconds. The culture inoculation, incubation and identification procedures were done under the supervision of a trained microbiologist. After vortexing, 50ìl of sample was plated onto selective culture media. Mitis-Salivarius agar(Himedia) was used for Streptococcus mitis, Mac Conkey agar(Himedia) was used for Enterococcus faecalis and Rogosa SL(Himedia) agar was used for Lactobacillus acidophilus. For culturing Streptococcus mitis a candle jar was used and incubated at 370C for 5 days. Aerobic culturing technique was used for Enterococcus faecalis and incubated at 370C for 2 days. Advanced anaerobic culturing technique using anaerobic glass jar and gaspak (Himedia) was used for Lactobacillus acidophilus and incubated at 370C for 7 days. After incubation, each plate was biochemically analysed for growth and identification of bacteria using the colony morphology and gram staining. Figure 2. Microbial culture technique STATISTICAL ANALYSIS : The data collected were typed onto a spreadsheet and statistically analysed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The results were statistically evaluated using Student unpaired T test and Pearson chi-square test. RESULTS : The study population age ranged from 20 to 70 years. Primary infection(mean age = 36 years) and secondary infection(mean age = 32 years) groups included equal proportion of men and women. Unpaired 't' test showed no significant difference between the groups in terms of age and gender distribution. In the present study, microorganisms were isolated from 73.3%(44/60) of selected teeth microbiologically sampled from primary and secondary infection by culture method.There were 26.7% of teeth(16/60) with negative culture on the plates. Primary infection yielded a total of 26 isolates. Streptococcus mitis was the most predominant microorganism isolated from 53.3% of primary root canal infection followed by Enterococcus faecalis (20%) and Lactobacillus acidophilus (13.3%).Chi-square test showed statistically significant association between Streptococcus mitis and primary infection (p= 0.018) as shown in Table 1. In secondary infection a total of 24 isolates were recovered. Enterococcus faecalis (46.7%) was the most frequently isolated microorganism followed by Streptococcus mitis (20%) and Lactobacillus acidophilus (13.3%). TABLE 1: Prevalence of Streptococcus mitis, Lactobacillus acidophilus and Enterococcus faecalis in patients with primary and secondary infections DISCUSSION : Our findings indicate that amongst the isolates, Streptococcus mitis(53.3%); a facultative anaerobic gram-positive species were predominant in infected root canals, which is corroborating with the studies of Sundqvist et al. and Molander et al. in which they also found facultative anaerobic and gram-positive species were predominant in primary infected root canals.[10] In contrast Baumgartner et al. and Gomes et al. have revealed the composition of necrotic pulp presents a polymicrobial flora characterized by a wide variety of combinations of bacteria, averaging 4-7 species per canal, predominantly anaerobic, with approximately equal proportions of Gram-negative and-positive bacteria.[11] Most of the previous studies have used non-selective culture methods. However,the present study did not evaluate gram negative microorganisms and it is noteworthy that the most prevalent species in primary infections may vary from study to study, which can be explained by several factors such as the sensitivity and specificity of the identification method, sampling technique, geographic location, and accuracy or divergence in clinical diagnosis.[12] In the present study Streptococcus mitis was significantly associated with primary root canal infections. Nisengard et al. has specified that the use of Mitis Salivarius agar helps in cultivation of only Streptococcus mitis suppressing the growth of other microorganisms.[13] Our study is in accordance with the study of Ercan et al. who also obtained Streptococcus mitis as one of the prevalent microorganism in primary root canal infections.[11] Moreover, Studies by Chavez et al. have revealed that Streptococci are present in high prevalence in infection of endodontic origin, including both chronic and acute periradicular diseases.[14] Enterococcus faecalis was isolated from 46.7% of retreatment University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 08 University J Dent Scie 2016; No. 2, Vol. 1 cases in the present investigation, this is in line with the findings of Molander et al.(47%) , Peciuliene et al.(38%) , Pinheiro et al. (55%) in which E. faecalis was the species most frequently isolated from retreatment cases.[1] This indicates the importance of this resistant microorganism in root canal treatment failure. The results of the present study showed that Enterococcus faecalis was isolated from 20% of primary root canal infection. Stuart et al. by using culture method have concluded that Enterococci constitute a small percentage of the microbial species isolated from root canals of teeth with necrotic dental pulps.[15] Mac Conkey agar is a specific media for definitive isolation of Enterococcus faecalis. However, PCR studies by Gomes et al. demonstrated E. faecalis to be as frequent in teeth with necrotic pulp as in teeth with failing endodontic treatment.[16] E. faecalis was present in 18% of the cases of primary endodontic infections as detected by Rocas et al. using nested PCR.[17] This raises the suspicion that the involvement of E. faecalis with failed cases can be a result of persistent infections. Based on this assumption, E. faecalis would take advantage of elimination of inhibitory members of the endodontic microbiota, or of its ability to resist intracanal procedures of disinfection.[18] Although the occurrence of E. faecalis in root-filled teeth associated with periradicular lesions can be presumed to be a result of persistent infections, the possibility of participation of this microorganism in secondary infections should not be disregarded. For the latter to occur, the microorganism should gain entry into the root canal during treatment, between appointments, or even after conclusion of the treatment. The origin of E. faecalis infecting root-filled teeth is worth elucidation for future research. Lactobacillus acidophilus was isolated from 13.3% of primary infection and retreatment cases. There was no statistical significant association between Lactobacillus acidophilus isolated from primary and secondary infection. This result is similar to Chavez et al. who found that Lactobacillus acidophilus are normal inhabitants of plaque and carious lesions but are not frequently identified in root canal infections.[19] Ercan et al. also isolated Lactobacillus from only 7.1% of infected root canals.[11] The fastidious and anaerobic nature of Lactobacillus sp. makes cultivation of this microorganism very difficult.[19] Further a brief exposure to air during the sampling procedure might be sufficient to kill anaerobic microorganisms.[20] In our study 26.7% of evalauated sample had no cultivable microorganism. Successful cultivation relies on viable microorganisms that live and grow on culture plates. The finding that 26.7% of root canals had no cultivable bacteria was not entirely unexpected; earlier studies were also unable to isolate bacteria from 55.6%(Sundqvist et al.), 26.6% (Molander et al.) and 17.5% (Peciuliene et al 2001) of teeth.[21] The present study evaluated only three microorganism, other microorganisms may also be associated with root canal infection. However, failure to detect bacteria does not prove their absence. Although the sampling techniques and laboratory procedures used in this study have been shown to be highly effective, it is indeed a technique sensitive method.[22] It is possible that some microorganisms present could have been lost, especially if the number of microorganisms present in the root canal was very low or if they were present in areas such as anatomical branches and apical areas obliterated by previous treatment. Furthermore, some microorganisms could have been removed together with the previous root filling and debris.[23] Follow-up studies on the root canal treatment report success of retreatment of teeth with apical periodontitis is lower, with an overall success rate of 66%.3 Molander et al. suggested that this poorer prognosis in root canal retreatments may be associated with difficulties in the elimination of the particular microflora in cases of root canal treatment failure.[24] Facultative anaerobic and Gram-positive bacteria, such as Enterococcus, Streptococcus are more resistant to instrumentation and to antiseptic agents, and therefore can be expected to persist more frequently in the root canal after inadequate root canal preparation and obturation.[19] Persisting microorganisms or their products can maintain an infectious process and cause treatment failure.[10] Hence, thorough knowledge of this microflora could guide new strategies to combat infection, leading to a better prognosis for root canal retreatments. CONCLUSION : Within the parameters of this study it can be concluded that Streptococcus mitis was most predominant in primary endodontic infection whereas Enterococcus faecalis was most frequently isolated from retreatment cases. This suggests that there is an affinity for specific microorganisms to be associated with specific type of endodontic infection. Accurate knowledge of the occurrence of the major putative endodontic pathogens and their implication in pathogenesis of periradicular diseases has the potential to afford subsidies for development of antimicrobial strategies effective in treating both primary and secondary infection. REFERENCES : 1. Figdor.D, Sundqvist.G. A big role for the very smallunderstanding the endodontic microbial flora. Aust Dent J 2007;52(1):38-51. 2. Siqueira J.F and Rocas I.N. Distinctive features of the microbiota associated with different forms of apical periodontitis. J Oral Microb 2009;1:22-44. 3. Aqrabawi J.A. Outcome of endodontic treatment of teeth University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 09 University J Dent Scie 2016; No. 2, Vol. 1 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. filled using lateral condensation versus vertical compaction. J Contem Dent Pract 2006;7(1):1-6. Nayak M, Kotigadde S, Shetty H, Vineet R V, Antony B. Impact Of Peptostreptococcus On Type 2 Diabetes Mellitus Related Secondary Root Canal Infections. Int J Pharm Sci Res 2013;4:4001-4009 Sundqvist.G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1994;78:522-530 Martin F.E,Nadkarni M.A,Jacques N.A,Hunter N. Quantitative microbiological study of human carious dentine by culture and real-time PCR:association of anaerobs with histopathological changes in chronic pulpitis. J Clin Microbiology 2002;40:1698-1704. Fouad A.F, Barry.J, Caimano.M, Clawson.M, Zhu.Q, Carver.R et al. PCR-based identification of bacteria associated with endodontic infections. J Clin Microb 2002;40(9):3223–3231. Bagg J, MacFarlane T. W, Poxton I.R, Smith A.J. Essentials of Microbiology for Dental Students. 2nd ed.United Kingdom: Oxford University Press;2006. Skucaite.N,Peciuliene.V,Vitkauskiene.A, Machiulskien e.V.Susceptibility of Endodontic pathogens to antibiotics in patients with symptomatic apical periodontitis. J Endod 2010;36:1611-1616 E s r a f i l B . G , A g h a z a d e h . M , A b a s h o v. R , M i l a n i A.S,Moosavi.Z. Microbial flora of root canals of pulpally-infected teeth:Enterococcus faecalis a prevalent species. J Dent Res Dent Clin Dent Pros 2009;3:24-27. Ercan.E,Dalli.M,Yavuz.L,Ozekinci.T.Investigation of microorganisms in infected dental root canals. Biotechnol.&Biotechnol 2006;20(2):166-172. Schirrmeister J.F, Liebenow A.L, Braun.G ,Wittmer.A , Hellwig.E, Ahmad.A Detection and eradication of microorganisms in root-filled teeth associated with periradicular lesions:an in-vivo study. J Endod 2007;33:536-540. Nisengard R.J, Newman M.G. Oral microbiology and immunology. 2nd ed. Philadelphia: W.B Sauders Co;1994. Chavez L.D, Svensater.G, Dahlen.G, Bergenholtz.G. Streptococci from root canals in teeth with apical periodontitis receiving endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:232241. Stuart C.H,Schwartz S.A,Beeson T.J,Owatz C.B.Enterococcus faecalis:Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-98. Gomes B.P, Pinheiro E.T, Sousa E.L, Jacinto R.C, Zaia.A, Ferraz C.R et al. Enterococcus faecalis in dental root canals detected by culture and by polymerase chain reaction analysis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:247-253. 17. Rocas I.N, Siqueira J.F and Santos R.N. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30(5):315-320. 18. Kuzekanani.M,Moaddab.S.Isolation of Enterococcus faecalis in previously root-filled canals in Kerman population.Dent news 2006;13:33-36. 19. Chavez L.D. Gram positive organisms in endodontic infections. Endod Topics 2004; 9: 79–96. 20. Mindere.A, Kundzina.R, Nikolajeva.V, Eze.D, Petrina.Z. Microflora of root filled teeth with apical periodontitis in Latvian patients. Stomatol,Baltic Dent and Maxillofacial J 2010;12:116-121. 21. Gomes B.P, Pinheiro E.T, Gade C.R, Souza E.L, Ferraz C.C, Zaia A.A et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71-76. 22. Spratt D.A. Significance of bacterial identification by molecular biology methods. Endod Topics 2004; 9: 5–14. 23. Pinheiro E.T, Gomes B.P, Ferraz C.C, Sousa E.L, Teixeira F.B & Filho F.J. Microorganisms from canals of root-filled teeth with periapical lesions.Int Endod J 2003;36:1-11. 24. Siqueira J.F. Aetiology of root canal treatment failure:why well-treated teeth can fail. Int Endod J 2001;34:1-10. CORRESPONDENCE AUTHOR: Dr. Vineet R. V. Department of Conservative Dentistry & Endodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari District, Tamil Nadu, India. Email: [email protected] University Journal of Dental Sciences, An Official Publication of Aligarh Muslim University, Aligarh. India 10