Clinical Research
Isolation of Alkaline-tolerant Bacteria from Primary Infected Root Canals Hui Pau Lew, DDS,*† Samantha Yiling Quah, MSc,* Jeen Nee Lui, BDS, MDS,† Gunnar Bergenholtz, DDS, PhD,‡ Victoria Soo Hoon Yu, BDS, MSc,* and Kai Soo Tan, PhD* Abstract Introduction: Alkaline-tolerant bacteria in primary infected root canals could have enhanced survival capacity against antimicrobials commonly used in root canal treatment. The aims of this study were to isolate and characterize alkaline-tolerant bacteria before endodontic treatment (S1), after chemomechanical root canal preparation (S2), and after calcium hydroxide dressing (S3). Methods: Bacteriologic samples were obtained from 43 primary infected root canals. Samples were inoculated into culture media at a pH of 9 and incubated anaerobically. The identities of bacterial isolates were determined by 16S ribosomal RNA sequencing. Results: All S1 samples were culture positive, with 70% harboring bacteria tolerating a pH of 9. Gram-positive bacteria Pseudoramibacter alactolyticus and Streptococcus spp were the most frequently isolated strains with a prevalence of 54%. Of 13 culture-positive S2 samples, 8 isolates tolerated a pH of 9, namely Streptococcus sanguinis, Enterococcus faecalis, Enterobacter cancerogenus, Streptococcus oralis, and Fusobacterium nucleatum. Seven of these 8 isolates (88%) were correspondingly isolated at S1. All 3 culture-positive S3 samples tolerated a pH of 9, namely S. sanguinis and E. faecalis, which were also isolated in the corresponding S1 and S2 samples. Conclusions: We showed that the presence of alkaline-tolerant Streptococcus and Enterococcus spp in primary infected root canals could lead to their persistence during and after root canal treatment and could pose a challenge to current treatment efficacy. (J Endod 2015;-:1–6)
Key Words Alkaline-tolerant bacteria, endodontic treatment, root canal infection
From the *Faculty of Dentistry, National University of Singapore, Singapore, Singapore; †National Dental Center, Singapore, Singapore; and ‡The Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden. Address requests for reprints to Dr Kai Soo Tan, Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083. E-mail address: denkst@nus. edu.sg 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.12.003
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radication of bacteria from the root canal system of teeth is an important goal of root canal therapy. Although radiographic healing was significantly better when no bacteria were recovered before filling (1, 2), studies have shown that mechanical instrumentation alone may reduce but not completely eliminate the microorganisms that reside in infected root canals (3, 4). Therefore, the use of irrigants and intracanal dressings with antimicrobial properties has been regarded as indispensable to endodontic therapy. Yet, even with sodium hypochlorite irrigation and calcium hydroxide (Ca[OH]2) intracanal medication, bacteria-free root canals are not always attained (5–8). The broad, nonspecific antibacterial effect of Ca(OH)2 comes from its high pH of 12.5, and its efficacy depends on maintaining this high pH to release hydroxyl ions during application. The availability of hydroxyl ions is influenced by the vehicle, additives, and dentin buffering effect. It has been reported in a recent study that pure Ca(OH)2 aqueous paste and Calasept (Scania Dental Ab, Knivsta, Sweden) maintained alkalinity better than viscous gel products such as DS Ca(OH)2 gel, Ultracal XS (Ultradent Products Inc, South Jordan, UT), Biokalkki (Dental Systems Oy, Helsinki, Finland), and Calxyl blue (OCO Pr€aparate GMBH, Dirmstein, Germany) when challenged with acid. However, these products were neutralized by dentin powder within 24 hours, reducing their antimicrobial effects (9). Along with the observation that the clinical outcome of single-visit endodontic treatment is comparable with that of multiple-visit treatments (10, 11), the singlevisit treatment approach has gained popularity in recent years. A drawback of singlevisit endodontic treatment is the potential for root canals to be left with residual bacteria and run the risk of treatment failure. Recently, a histobacteriologic investigation comparing the effectiveness of Ca(OH)2 in single- and 2-visit endodontic treatment showed that interappointment medication with Ca(OH)2 significantly reduced the bacterial load in ramifications, isthmuses, and dentinal tubules. However, bacteria-free root canals were not consistently achieved, and long-term follow up data were not reported (12). One way forward is to characterize the types of root canal bacteria that are able to withstand endodontic treatment procedures. We hypothesize that bacterium with the ability to tolerate an alkaline environment, at which common antimicrobial irrigants and dressing operate, have a survival edge that may allow them to persist in the various stages of root canal treatment. It is reasonable to assume that the colonization of root canals with alkaline-tolerant microorganisms can lead to a higher probability of such microorganisms surviving chemomechanical procedures. Some conditions can enhance the ability of bacteria to survive exposure to alkaline treatments. Br€andle et al (13) studied the impact of growth conditions on the susceptibility of 5 taxa of microbes to alkaline stress. They found that planktonic Enterococcus faecalis and Candida albicans could withstand Ca(OH)2 exposure up to 100 minutes. Dentin adhesion greatly enhanced the resistance of E. faecalis and Actinomyces naeslundii to Ca(OH)2, whereas multispecies biofilm coaggregates enhanced the resistance of Streptococcus sobrinus. Nakajo et al (14) isolated 29 alkaline-resistant bacterial species from 37 primarily infected teeth. The authors reported that the predominant alkaline-resistant bacteria belonged to the Enterococcus genus. Their report obtained bacteria from teeth that were extracted because of severe root canal infection. Therefore, their results only describe bacteria present at this end stage. Furthermore, their methods did not allow the study of bacteria from different stages of endodontic treatment.
Isolation of Alkaline-tolerant Bacteria
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Clinical Research In this study, we sought to isolate alkaline-tolerant bacteria before endodontic treatment (S1), after chemomechanical root canal preparation (S2), and after the placement of an interappointment Ca(OH)2 dressing (S3).
Materials and Methods Selection Criteria A total of 50 patients were recruited in this study. Ethics approval was obtained from the Domain Specific Review Board, National Healthcare Group. Informed consents were obtained from all patients. Included were teeth with pulp necrosis, clinical and radiographic evidence of periapical periodontitis, sufficient tooth structure for adequate isolation, a periodontal pocket depth of less than 4 mm, and no history of previous endodontic treatment. Patients who had received antibiotic therapy 3 months before treatment or suffered from immunocompromised disease and cracked teeth were excluded. Of the 50 cases, 43 passed the sterility control check. These teeth consisted of 15 anterior teeth, 8 premolars, and 20 molars. A total of 23 symptomatic (tenderness to percussion only) and 20 asymptomatic cases were examined. Sampling Procedures One endodontist and 9 full-time residents enrolled in the Postgraduate Endodontics Residency Program were trained and performed the same sampling procedures. A written checklist and a data collection form were used to ensure consistency among operators. Briefly, teeth were cleaned, and care was taken not to encroach into the pulp chamber during caries or restoration removal. Significant losses of tooth structure were rebuilt to secure leakage-free sampling conditions. After rubber dam isolation, dental floss was tightened around the cervical region. The operation field and tooth were sterilized using 30% hydrogen peroxide and 10% iodine tincture (15). Iodine tincture was inactivated with 5% sodium thiosulfate before a sterility check performed by scrubbing a cotton pellet against the tooth surface where the access opening was to be performed. The cotton pellet was aseptically transferred to a sterile tube containing thioglycollate broth (Sigma-Aldrich, St Louis, MO) and incubated anaerobically for 5 days. Samples that failed the sterility check were excluded from the study. An access opening to the pulp chamber was prepared with a sterile bur. A small amount of reduced transport fluid (RTF) (16) was added to the canal. The canal was instrumented with a sterile stainless steel #15 K-file 1 mm short of the radiographic apex (estimated from the diagnostic radiograph) to release dentinal shavings. The smeared fluid in the canal was absorbed with 5 successively applied paper points, with each paper point left in the canal for at least 30 seconds. This bacteriologic sample, termed S1, was placed in a sterile Eppendorf tube containing 250 mL RTF. The tube was immediately transported to the laboratory for microbiological analysis. After coronal preflaring and working length determination, the canal was instrumented sequentially with hand/rotary files to the desired master apical file size. Canal irrigation with 1.25% sodium hypochlorite was delivered with a 5-mL disposable syringe and a 27-G needle after each instrumentation. After completion of this treatment sequence, the remaining sodium hypochlorite was neutralized with 5% sodium thiosulfate. The canal was dried with paper points, and RTF was added. A sterile Kfile 1 size larger than the master apical file was inserted to the working length and rotated to release dentinal shavings. The K-file was removed, cut off at the apical 5 mm, and transferred to the second RTF vial according to the technique designed by Ørstavik et al (4). The remaining RTF was absorbed with 5 paper points and was transferred to the same RTF vial as the cut K-file. This sample served as the postinstrumentation sample (S2). In all cases, the root canal instrumentation was completed 2
Lew et al.
during the first appointment. After these procedures, a slurry paste of Ca(OH)2 mixed with sterile water was placed into the canal with a Lentulo spiral, and the access cavity was dressed with Intermediate Restorative Material (IRM; Dentsply Caulk, Milford, DE). Ca(OH)2 dressing was left in the canal for at least 1 week. During the second appointment, a sterility control sample was obtained. The IRM dressing was removed aseptically under rubber dam isolation. The intracanal Ca(OH)2 was removed with ultrasonically agitated irrigation of the physiological saline. Inspection with microscopic magnification confirmed the removal of Ca(OH)2. A third bacteriologic sample (S3) was then taken according to the procedure described for the S2 sample, except that a K-file 2 sizes larger than the master apical file was used. After sampling, the canal was dried and filled with gutta-percha and sealer.
Monitoring of pH under Culture Conditions The pH of the brain-heart infusion (BHI) broth (14) (Acumedia, Lansing, MI) supplemented with yeast extract (Acumedia), hemin (Sigma-Aldrich), and vitamin K (Sigma-Aldrich) was adjusted to a pH of 9 with 10 mol/L potassium hydroxide (Sigma-Aldrich) using a pH meter and incubated at 37 C anaerobically. Changes in pH were monitored daily up to a period of 5 days. Experiments were performed 3 times and each time in triplicate. Bacterial Culture Samples were vortexed to dislodge bacteria from the paper points. The amount of bacteria present in the root canal sample was determined by serial dilution in BHI broth and plating on Tryptic soy agar (TSA) supplemented with 5% sheep blood agar (Oxoid, Hampshire, UK). A 50-mL bacterial suspension was inoculated into 3 mL buffered BHI broth supplemented with yeast extract, hemin, and vitamin K at a pH of 9 and incubated at 37 C for 3 days. Cultures were incubated at 37 C in a DG250 Anaerobic Workstation (Don Whitley Scientific, West Yorkshire, UK) supplied with 80% N2, 10% H2, and 10% CO2. Uninoculated (pH = 9) adjusted media served as an aseptic control. If turbidity was observed after 3 days of culture, the bacterial culture was subcultured at a ratio of 1:20 into a tube containing 2 mL BHI broth supplemented with yeast extract, hemin, and vitamin K with the pH adjusted to 9 to further confirm that the bacteria were capable of growing under alkaline conditions. When turbidity was observed, the bacterial suspension was streaked on a TSA sheep blood agar plate. Identification of Bacterial Isolates Isolated colonies, which were monocultures at this point, were inoculated into 2 mL buffered BHI broth supplemented with yeast extract, hemin, and vitamin K at a pH of 9 and incubated anaerobically at 37 C. Genomic DNA was extracted using the QiaAmp Minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) using a universal primer set (forward, 50 -GAGAGTTTGATYMTGGCTCAG; reverse, 50 -GAAGGAGGTGWTCCARCCGCA) (17). A PCR mixture consisted of a 50-ng template of DNA, 20 pmol each of forward and reverse primers, and 2 GoTaq PCR mastermix (Promega, Madison, WI) in a final reaction volume of 100 mL. The following thermal cycling conditions were used: initial denaturation at 94 C for 3 minutes, denaturation at 94 C for 30 seconds, annealing at 58 C for 30 seconds, and extension at 72 C for 1 minute 45 seconds. A final extension at 72 C for 10 minutes was performed. PCR was performed for 25 cycles in an iCycler (BioRad, Hercules, CA). An aliquot of the PCR product was analyzed by electrophoresis on a 1% agarose gel. The PCR product was purified using Wizard SV Gel and the PCR Clean Up System (Promega) according JOE — Volume -, Number -, - 2015
Clinical Research to the manufacturer’s protocol. The PCR product, which was approximately 1.5 kb in size, was sequenced by capillary sequencing (AIT Biotech, Singapore, Singapore). For identification, DNA sequences were compared with the 16S ribosomal RNA sequences in the Human Oral Microbiome Database. A cutoff of value of 98.5% identity was set (18).
Assessment of pH Tolerance of Bacterial Isolates Bacterial colonies were inoculated into 2 mL buffered BHI broth supplemented with yeast extract, hemin, and vitamin K with pH levels adjusted to 9, 10, and 11 with 10 mol/L potassium hydroxide and incubated anaerobically at 37 C for 3 days. The amount of bacterial growth was measured by optical density at 600 nm. The relationship between optical density and colony-forming units was determined for each species by serial dilution and plating on TSA plate sheep blood agar plates. Based on optical density values, bacterial growth was categorized as none (), weak (+), moderate (++), or heavy (+++).
Results Tests were performed to determine the sustainability of alkaline pH under the culture conditions. As shown in Figure 1, in the absence of bacteria, alkaline pH could be maintained within the range of 9.0 0.2 pH units for a maximum period of 3 days. All 43 samples were culture positive at S1 with amounts ranging from 102–107 colony-forming units as determined by anaerobic culture. A total of 30 samples (70%) were found to harbor alkaline-tolerant bacteria (Table 1). Consistently only 1 alkaline-tolerant bacterial species was isolated from each S1 sample. Among the bacterial isolates obtained, 23 of 30 (77%) were gram-positive bacteria (Table 2), with Pseudoramibacter alactolyticus and Streptococci spp being the most frequently isolated with a prevalence of 54%. The other bacterial isolates obtained were Enterobacter cancerogenus, Fusobacterium nucleatum, and Enterococcus faecalis. Among the 13 culture-positive S2 samples, 8 alkaline-tolerant bacteria were isolated. There was a correlation between the presence of these organisms at S1 and their persistence at S2. Seven of 8 bacterial isolates (88%) retrieved in S2 samples were correspondingly obtained at S1. Streptococcus spp, E. faecalis, E. cancerogenus, and F. nucleatum were among the bacterial isolates obtained at S2. Among the 3 culture-positive samples at S3, all possessed alkaline-tolerant isolates,
Figure 1. pH changes of culture media over time. BHI broth supplemented with yeast extract, hemin, and vitamin K was adjusted to pH 9 and incubated at 37 C anaerobically in the absence of bacteria. pH was taken daily for up to 5 days.
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namely Streptococcus sanguinis and E. faecalis. These species were also isolated from the corresponding S1 and S2 samples. To determine the degree of alkaline tolerance of the bacterial isolates, the growth of these bacteria were further assessed at pH levels of 10 and 11. E. faecalis was the only bacterial species capable of surviving a pH of 11, whereas E. cancerogenus was able to withstand a pH of 10 (Table 3).
Discussion Few studies have addressed the issue of alkaline tolerance among root canal bacteria. In the 2 other studies reported so far, both described the predominance of gram-positive bacteria enduring alkaline conditions, with biofilm forms being more resistant than planktonic cells (14, 19). In this study, we showed that more than two thirds of infected root canal samples harbored alkaline-tolerant bacteria; gram-positive bacteria dominated. The first stage of sampling at S1 began from untreated necrotic pulps to isolate alkaline-tolerant bacteria from the initial colonizing population in the root canal. Thus, isolates at this stage represent bacteria that have not been exposed to alkaline stress but have the inherent ability to grow under laboratory conditions at a pH of 9 even without adaptation from pre-exposure to alkaline medicaments. P. alactolyticus and Streptococcus spp were the most frequently isolated species, whereas E. faecalis occurred only in 2 cases. The differences to the findings of Nakajo et al (14) may be caused by the sampling method adopted in that study in which bacteria from infected root canals were obtained from homogenized dentin powder from extracted teeth. Our results further showed that when alkaline-tolerant bacteria were found in infected root canals, only 1 bacterial isolate was obtained from each canal. The reason behind this observation, although interesting, is currently unclear. This may be attributed to the fact that bacteria surviving in the root canal are present in biofilm communities where alkaline-tolerant microorganisms may produce bacteriocins to suppress the growth of competing bacterial species (20). P. alactolyticus is a strict anaerobic gram-positive rod. Several studies have shown this species to be a common resident of primary root canal infections with a prevalence ranging from 34%–76% (21–23). In the present study, P. alactolyticus was the most frequently isolated bacteria at S1. However, this bacterial species was not obtained from subsequent S2 and S3 samples. Our observations agree with data from a previous study that showed that P. alactolyticus is easily eliminated by endodontic disinfection procedures (24). Our results show that P. alactolyticus, although culturable at a pH of 9, was not able to withstand the higher pH of sodium hypochlorite irrigation and mechanical debridement. This organism may nevertheless play a significant role in the pathogenesis of primary root canal infections. Streptococcus spp was as equally prevalent as P. alactolyticus in S1 samples. Of the 8 isolates obtained at S1, 4 persisted in S2 samples and 2 in S3 samples. Streptococcus sanguinis and Streptococcus oralis appeared to be the most resilient species obtained in this study, showing a high resistance toward instrumentation and antimicrobial irrigants and/or Ca(OH)2 dressing. In a study investigating the diversity of Streptococcus spp in root canals undergoing endodontic therapy, S. gordonii and S. oralis were the most prevalent (25). The presence of gram-positive bacteria, namely Lactobacillus spp, the nonmutans group Streptococci (S. sanguinis, S. gordonii, S. oralis, and Streptococcus mitis), and Enterococcus spp, have been found to be significantly correlated with cases that had undergone chemomechanical procedures and interappointment medicaments (26). These differences may be attributed to variations associated with the patient pool.
Isolation of Alkaline-tolerant Bacteria
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Clinical Research TABLE 1. Alkaline-tolerant Bacterial Species at S1, S2, and S3 Case No
Bacterial species at S1
A43 A45 A49 A50 A54 A61 A63 A65 A67 A71 A79 A90 A94 A97 A100 A116 A124 A127 A143 A144 A146 A148 A150 A154 A157 A158 A159 A162 A179 A181
Bacterial species at S2
Parvimonas micra Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Enterococcus faecalis Enterobacter cancerogenus Fusobacterium nucleatum Pseudoramibacter alactolyticus Klebsiella pneumonia Parvimonas micra Streptococcus oralis Pseudoramibacter alactolyticus Peptostreptococcus stomatis Streptococcus sanguinis Propionibacterium propionicum Streptococcus sanguinis Peptostreptococcus stomatis Fusobacterium nucleatum Streptococcus intermedius Fusobacterium nucleatum Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Enterobacter cancerogenus Streptococcus sanguinis Streptococcus intermedius Enterobacter cancerogenus Streptococcus sanguinis Streptococcus oralis Enterococcus faecalis
Bacterial species at S3
Enterococcus faecalis Enterobacter cancerogenus
Fusobacterium nucleatum Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus sanguinis Streptococcus oralis Enterococcus faecalis
Enterococcus faecalis
Bacterial counts (cfu/mL) at S1 6 106 2 102 1.6 104 2.2 105 1 105 5.8 107 8 106 5 105 1.3 103 1 107 3.38 107 2.62 107 1.12 105 8 104 7 106 1.82 104 2 106 4.14 106 2.4 102 1.64 106 1.2 105 6.2 104 4 104 2 104 1.6 104 1.02 105 1.74 103 1.2 103 1 104 1.12 107
Case number refers to bacterial species of the respective root canal samples that have been obtained by culturing the samples at a pH of 9. S1, S2, and S3 refer to bacterial species obtained before endodontic treatment, after chemomechanical root canal preparation, and after placement of calcium hydroxide dressing, respectively.
In our sample population, Streptococci and Enterococci spp were found to be the predominant alkaline-tolerant bacterial types that survived chemomechanical treatment and the placement of Ca(OH)2 dressing. These observations may be attributed to the organisms’ ability to overcome stresses associated with root canal treatment such as antimicrobial usage, changes in redox potential, and changes in nutrients (27). As expected, the bacterial load was greatly reduced after chemomechanical treatment. After irrigation and mechanical instrumentation (S2), 8 of 30 samples (27%) with alkaline-tolerant organisms in S1
TABLE 2. Occurrence of Alkaline-tolerant Bacterial Species Bacterial species Gram-positive rod Pseudoramibacter alactolyticus Propionibacterium propionicum Gram-positive cocci Enterococcus faecalis Streptococcus sanguinis Streptococcus intermedius Streptococcus oralis Peptostreptococcus stomatis Parvimonas micra Gram-negative rod Fusobacterium nucleatum Enterobacter cancerogenus Gram-negative cocci Klebsiella pneumonia Total
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Lew et al.
S1
S2
S3
2 4 2 2 2 2
2 3
1 2
3 3
1 1
1 30
8
8 1
1
3
were recovered. Alkaline-tolerant bacteria isolated at S2 included 4 isolates of Streptococcus spp, 2 isolates of E. faecalis, and 1 isolate each of E. cancerogenus and F. nucleatum. Isolates obtained at the S2 stage represent bacteria that can withstand acute exposure to the high pH of sodium hypochlorite irrigation. The number of culture-positive samples obtained at S3 was 3 of 43 (7%), which is similar to the findings reported by Shuping et al (28). Isolates obtained at the S3 stage represent bacteria that withstood sodium hypochlorite irrigation as well as prolonged exposure to Ca(OH)2 dressing. These bacteria isolates either possess or have acquired robust mechanisms to survive alkaline conditions. Although the numbers obtained were relatively small, it is significant given that all 3 culture-positive samples harbored alkaline-tolerant organisms, namely S. sanguinis and E. faecalis. More importantly, the same organisms were isolated at the respective S1 and S2 stages, suggesting that alkaline-tolerant bacteria belonging to the genus of Streptococcus and Enterococcus may possess an enhanced ability to withstand endodontic treatment procedures. F. nucleatum was the only bacterial isolate obtained at S2 without appearing in the corresponding S1 sample. Possible explanations include culture reversal (6) and contamination from the oral environment during treatment or interappointment interval. Another possible explanation is that the cell count of F. nucleatum was low during the first sampling such that it was not isolatable, but the amounts increased in numbers in the canal to become retrievable after irrigation and instrumentation. Among the alkaline-tolerant bacteria obtained in this study, only E. cancerogenus and E. faecalis were able to persist at a pH $9, enduring a pH of 10 and 11, respectively. It has been reported in numerous studies that E. faecalis is capable of enduring high alkaline conditions JOE — Volume -, Number -, - 2015
Clinical Research TABLE 3. Degree of Alkaline Tolerance of Bacterial Species Bacterial species
pH 9
pH 10
pH 11
Enterococcus faecalis Enterobacter cancerogenus Fusobacterium nucleatum Pseudoramibacter alactolyticus Klebsiella pneumonia Streptococcus oralis Parvimonas micra Peptostreptococcus stomatis Propionibacterium propionicum Fusobacterium nucleatum Enterobacter cancerogenus Streptococcus sanguinis Streptococcus intermedius Peptostreptococcus stomatis Fusobacterium nucleatum
+++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
++ +
+
, no growth; +, weak growth; ++, moderate growth; +++, heavy growth.
up to a pH of 11 (29–31), likely through the action of proton pumps, which allow the bacteria to maintain intracellular pH homeostasis (32). The cellular mechanism behind E. cancerogenus’ ability to withstand a pH of up to 10 is currently unknown and awaits future studies. The survival of alkaline-tolerant bacteria at S2 and S3 in the present study may be attributed to the influence of dentin on the antimicrobial actions of sodium hypochlorite and Ca(OH)2. The time required for sodium hypochlorite to eradicate bacteria increased significantly if it had been preincubated with dentin powder (33). In a recent study, Macedo et al (34) further reported that the pH of unbuffered sodium hypochlorite solution dropped from a pH of 12 to a pH of 10.5 after 50 seconds of contact time with dentin. The antibacterial effects of Ca(OH)2 have been controversial because of inconsistencies in obtaining negative cultures from canals treated with Ca(OH)2 (6, 7, 8, 12, 29). Although this may be partially attributed to a lack of direct contact of microorganisms with Ca(OH)2 in the root canal, increasing evidence show that dentin is able to buffer the high pH of Ca(OH)2, which is essential for its antimicrobial action (33, 35). One of the limitations of this study was that not all bacteria obtained from sampling are isolatable. Although we selected a media that allowed the growth of as wide a range of bacteria as possible, bacteria with more stringent or unique culture requirements than those in this study would not be isolated. Alkaline pH was also not sustainable for more than 3 days under anaerobic conditions. The cultures were isolated no later than 3 days to avoid growth of other bacteria when the pH drops below 9. Thus, slower-growing potential alkaline bacteria would not be isolated. Another limitation of this study was the sampling method in which paper points were unable to reach the irregularities of the root canal system such as the fins, isthmuses, lateral canals, apical delta, and dentinal tubules. Hence, the alkaline-resistant bacteria isolated in this study were likely representative of the microflora in the main root canals. The current study was undertaken to characterize the relationship between the tolerance of bacterial isolates to a pH of 9 and survival. It remains to be determined if these bacterial isolates adopt universal physiological responses to endure high pH conditions. Such knowledge may allow us to develop enhanced antimicrobial approaches against these microorganisms.
Conclusions This study describes alkaline-tolerant species present at various stages of endodontic therapy. Although the mechanisms adopted by these alkaline-tolerant bacterial isolates remain to be JOE — Volume -, Number -, - 2015
determined, our results suggested that the presence of these bacteria in primary root canal infections could pose a challenge to current treatment efficacy.
Acknowledgments Supported by a grant (R221-000-051-112) from the Ministry of Education Singapore. The authors deny any conflicts of interests related to this study.
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intermedia, and Pseudoramibacter alactolytivus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:129–34. Chavez de Paz LE, Svens€ater 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;100:232–41. Chavez de Paz LE, Dahlen G, Molander A, et al. Bacteria recovered from teeth with apical apical periodontitis after antimicrobial endodontic treatment. Int Endod J 2003;36:500–8. Sundqvist G, Figdor D. Life as an endodontic pathogen. Endod Topics 2003;6:3–28. Shuping GB, Ørstavik D, Sigurdsson A, Trope M. Reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications. J Endod 2000;26:751–5. Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1–7.
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30. Sundqvist G, Figdor D, Persson S, Sj€ogren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86–93. 31. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J 2001;34:429–34. 32. Evans M, Davies JK, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int Endod J 2002;35:221–8. 33. Haapasalo HK, Siren EK, Waltimo TMT, et al. Inactivation of local root canal medicaments by dentine: an in vitro study. Int Endod J 2000;33:126–31. 34. Macedo RG, Herrero NP, Wesselink P, et al. Influence of the dentinal wall on the pH of sodium hypochlorite during root canal irrigation. J Endod 2014;40:1005–8. 35. Quah SY, Wu S, Lui JN, et al. N-Acetylcysteine inhibits growth and eradicates biofilm of Enterococcus faecalis. J Endod 2012;38:81–5.
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Isolation of Alkaline-tolerant Bacteria from Primary Infected Root Canals Hui Pau Lew, DDS,*† Samantha Yiling Quah, MSc,* Jeen Nee Lui, BDS, MDS,† Gunnar Bergenholtz, DDS, PhD,‡ Victoria Soo Hoon Yu, BDS, MSc,* and Kai Soo Tan, PhD* Abstract Introduction: Alkaline-tolerant bacteria in primary infected root canals could have enhanced survival capacity against antimicrobials commonly used in root canal treatment. The aims of this study were to isolate and characterize alkaline-tolerant bacteria before endodontic treatment (S1), after chemomechanical root canal preparation (S2), and after calcium hydroxide dressing (S3). Methods: Bacteriologic samples were obtained from 43 primary infected root canals. Samples were inoculated into culture media at a pH of 9 and incubated anaerobically. The identities of bacterial isolates were determined by 16S ribosomal RNA sequencing. Results: All S1 samples were culture positive, with 70% harboring bacteria tolerating a pH of 9. Gram-positive bacteria Pseudoramibacter alactolyticus and Streptococcus spp were the most frequently isolated strains with a prevalence of 54%. Of 13 culture-positive S2 samples, 8 isolates tolerated a pH of 9, namely Streptococcus sanguinis, Enterococcus faecalis, Enterobacter cancerogenus, Streptococcus oralis, and Fusobacterium nucleatum. Seven of these 8 isolates (88%) were correspondingly isolated at S1. All 3 culture-positive S3 samples tolerated a pH of 9, namely S. sanguinis and E. faecalis, which were also isolated in the corresponding S1 and S2 samples. Conclusions: We showed that the presence of alkaline-tolerant Streptococcus and Enterococcus spp in primary infected root canals could lead to their persistence during and after root canal treatment and could pose a challenge to current treatment efficacy. (J Endod 2015;-:1–6)
Key Words Alkaline-tolerant bacteria, endodontic treatment, root canal infection
From the *Faculty of Dentistry, National University of Singapore, Singapore, Singapore; †National Dental Center, Singapore, Singapore; and ‡The Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden. Address requests for reprints to Dr Kai Soo Tan, Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083. E-mail address: denkst@nus. edu.sg 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.12.003
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radication of bacteria from the root canal system of teeth is an important goal of root canal therapy. Although radiographic healing was significantly better when no bacteria were recovered before filling (1, 2), studies have shown that mechanical instrumentation alone may reduce but not completely eliminate the microorganisms that reside in infected root canals (3, 4). Therefore, the use of irrigants and intracanal dressings with antimicrobial properties has been regarded as indispensable to endodontic therapy. Yet, even with sodium hypochlorite irrigation and calcium hydroxide (Ca[OH]2) intracanal medication, bacteria-free root canals are not always attained (5–8). The broad, nonspecific antibacterial effect of Ca(OH)2 comes from its high pH of 12.5, and its efficacy depends on maintaining this high pH to release hydroxyl ions during application. The availability of hydroxyl ions is influenced by the vehicle, additives, and dentin buffering effect. It has been reported in a recent study that pure Ca(OH)2 aqueous paste and Calasept (Scania Dental Ab, Knivsta, Sweden) maintained alkalinity better than viscous gel products such as DS Ca(OH)2 gel, Ultracal XS (Ultradent Products Inc, South Jordan, UT), Biokalkki (Dental Systems Oy, Helsinki, Finland), and Calxyl blue (OCO Pr€aparate GMBH, Dirmstein, Germany) when challenged with acid. However, these products were neutralized by dentin powder within 24 hours, reducing their antimicrobial effects (9). Along with the observation that the clinical outcome of single-visit endodontic treatment is comparable with that of multiple-visit treatments (10, 11), the singlevisit treatment approach has gained popularity in recent years. A drawback of singlevisit endodontic treatment is the potential for root canals to be left with residual bacteria and run the risk of treatment failure. Recently, a histobacteriologic investigation comparing the effectiveness of Ca(OH)2 in single- and 2-visit endodontic treatment showed that interappointment medication with Ca(OH)2 significantly reduced the bacterial load in ramifications, isthmuses, and dentinal tubules. However, bacteria-free root canals were not consistently achieved, and long-term follow up data were not reported (12). One way forward is to characterize the types of root canal bacteria that are able to withstand endodontic treatment procedures. We hypothesize that bacterium with the ability to tolerate an alkaline environment, at which common antimicrobial irrigants and dressing operate, have a survival edge that may allow them to persist in the various stages of root canal treatment. It is reasonable to assume that the colonization of root canals with alkaline-tolerant microorganisms can lead to a higher probability of such microorganisms surviving chemomechanical procedures. Some conditions can enhance the ability of bacteria to survive exposure to alkaline treatments. Br€andle et al (13) studied the impact of growth conditions on the susceptibility of 5 taxa of microbes to alkaline stress. They found that planktonic Enterococcus faecalis and Candida albicans could withstand Ca(OH)2 exposure up to 100 minutes. Dentin adhesion greatly enhanced the resistance of E. faecalis and Actinomyces naeslundii to Ca(OH)2, whereas multispecies biofilm coaggregates enhanced the resistance of Streptococcus sobrinus. Nakajo et al (14) isolated 29 alkaline-resistant bacterial species from 37 primarily infected teeth. The authors reported that the predominant alkaline-resistant bacteria belonged to the Enterococcus genus. Their report obtained bacteria from teeth that were extracted because of severe root canal infection. Therefore, their results only describe bacteria present at this end stage. Furthermore, their methods did not allow the study of bacteria from different stages of endodontic treatment.
Isolation of Alkaline-tolerant Bacteria
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Clinical Research In this study, we sought to isolate alkaline-tolerant bacteria before endodontic treatment (S1), after chemomechanical root canal preparation (S2), and after the placement of an interappointment Ca(OH)2 dressing (S3).
Materials and Methods Selection Criteria A total of 50 patients were recruited in this study. Ethics approval was obtained from the Domain Specific Review Board, National Healthcare Group. Informed consents were obtained from all patients. Included were teeth with pulp necrosis, clinical and radiographic evidence of periapical periodontitis, sufficient tooth structure for adequate isolation, a periodontal pocket depth of less than 4 mm, and no history of previous endodontic treatment. Patients who had received antibiotic therapy 3 months before treatment or suffered from immunocompromised disease and cracked teeth were excluded. Of the 50 cases, 43 passed the sterility control check. These teeth consisted of 15 anterior teeth, 8 premolars, and 20 molars. A total of 23 symptomatic (tenderness to percussion only) and 20 asymptomatic cases were examined. Sampling Procedures One endodontist and 9 full-time residents enrolled in the Postgraduate Endodontics Residency Program were trained and performed the same sampling procedures. A written checklist and a data collection form were used to ensure consistency among operators. Briefly, teeth were cleaned, and care was taken not to encroach into the pulp chamber during caries or restoration removal. Significant losses of tooth structure were rebuilt to secure leakage-free sampling conditions. After rubber dam isolation, dental floss was tightened around the cervical region. The operation field and tooth were sterilized using 30% hydrogen peroxide and 10% iodine tincture (15). Iodine tincture was inactivated with 5% sodium thiosulfate before a sterility check performed by scrubbing a cotton pellet against the tooth surface where the access opening was to be performed. The cotton pellet was aseptically transferred to a sterile tube containing thioglycollate broth (Sigma-Aldrich, St Louis, MO) and incubated anaerobically for 5 days. Samples that failed the sterility check were excluded from the study. An access opening to the pulp chamber was prepared with a sterile bur. A small amount of reduced transport fluid (RTF) (16) was added to the canal. The canal was instrumented with a sterile stainless steel #15 K-file 1 mm short of the radiographic apex (estimated from the diagnostic radiograph) to release dentinal shavings. The smeared fluid in the canal was absorbed with 5 successively applied paper points, with each paper point left in the canal for at least 30 seconds. This bacteriologic sample, termed S1, was placed in a sterile Eppendorf tube containing 250 mL RTF. The tube was immediately transported to the laboratory for microbiological analysis. After coronal preflaring and working length determination, the canal was instrumented sequentially with hand/rotary files to the desired master apical file size. Canal irrigation with 1.25% sodium hypochlorite was delivered with a 5-mL disposable syringe and a 27-G needle after each instrumentation. After completion of this treatment sequence, the remaining sodium hypochlorite was neutralized with 5% sodium thiosulfate. The canal was dried with paper points, and RTF was added. A sterile Kfile 1 size larger than the master apical file was inserted to the working length and rotated to release dentinal shavings. The K-file was removed, cut off at the apical 5 mm, and transferred to the second RTF vial according to the technique designed by Ørstavik et al (4). The remaining RTF was absorbed with 5 paper points and was transferred to the same RTF vial as the cut K-file. This sample served as the postinstrumentation sample (S2). In all cases, the root canal instrumentation was completed 2
Lew et al.
during the first appointment. After these procedures, a slurry paste of Ca(OH)2 mixed with sterile water was placed into the canal with a Lentulo spiral, and the access cavity was dressed with Intermediate Restorative Material (IRM; Dentsply Caulk, Milford, DE). Ca(OH)2 dressing was left in the canal for at least 1 week. During the second appointment, a sterility control sample was obtained. The IRM dressing was removed aseptically under rubber dam isolation. The intracanal Ca(OH)2 was removed with ultrasonically agitated irrigation of the physiological saline. Inspection with microscopic magnification confirmed the removal of Ca(OH)2. A third bacteriologic sample (S3) was then taken according to the procedure described for the S2 sample, except that a K-file 2 sizes larger than the master apical file was used. After sampling, the canal was dried and filled with gutta-percha and sealer.
Monitoring of pH under Culture Conditions The pH of the brain-heart infusion (BHI) broth (14) (Acumedia, Lansing, MI) supplemented with yeast extract (Acumedia), hemin (Sigma-Aldrich), and vitamin K (Sigma-Aldrich) was adjusted to a pH of 9 with 10 mol/L potassium hydroxide (Sigma-Aldrich) using a pH meter and incubated at 37 C anaerobically. Changes in pH were monitored daily up to a period of 5 days. Experiments were performed 3 times and each time in triplicate. Bacterial Culture Samples were vortexed to dislodge bacteria from the paper points. The amount of bacteria present in the root canal sample was determined by serial dilution in BHI broth and plating on Tryptic soy agar (TSA) supplemented with 5% sheep blood agar (Oxoid, Hampshire, UK). A 50-mL bacterial suspension was inoculated into 3 mL buffered BHI broth supplemented with yeast extract, hemin, and vitamin K at a pH of 9 and incubated at 37 C for 3 days. Cultures were incubated at 37 C in a DG250 Anaerobic Workstation (Don Whitley Scientific, West Yorkshire, UK) supplied with 80% N2, 10% H2, and 10% CO2. Uninoculated (pH = 9) adjusted media served as an aseptic control. If turbidity was observed after 3 days of culture, the bacterial culture was subcultured at a ratio of 1:20 into a tube containing 2 mL BHI broth supplemented with yeast extract, hemin, and vitamin K with the pH adjusted to 9 to further confirm that the bacteria were capable of growing under alkaline conditions. When turbidity was observed, the bacterial suspension was streaked on a TSA sheep blood agar plate. Identification of Bacterial Isolates Isolated colonies, which were monocultures at this point, were inoculated into 2 mL buffered BHI broth supplemented with yeast extract, hemin, and vitamin K at a pH of 9 and incubated anaerobically at 37 C. Genomic DNA was extracted using the QiaAmp Minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) using a universal primer set (forward, 50 -GAGAGTTTGATYMTGGCTCAG; reverse, 50 -GAAGGAGGTGWTCCARCCGCA) (17). A PCR mixture consisted of a 50-ng template of DNA, 20 pmol each of forward and reverse primers, and 2 GoTaq PCR mastermix (Promega, Madison, WI) in a final reaction volume of 100 mL. The following thermal cycling conditions were used: initial denaturation at 94 C for 3 minutes, denaturation at 94 C for 30 seconds, annealing at 58 C for 30 seconds, and extension at 72 C for 1 minute 45 seconds. A final extension at 72 C for 10 minutes was performed. PCR was performed for 25 cycles in an iCycler (BioRad, Hercules, CA). An aliquot of the PCR product was analyzed by electrophoresis on a 1% agarose gel. The PCR product was purified using Wizard SV Gel and the PCR Clean Up System (Promega) according JOE — Volume -, Number -, - 2015
Clinical Research to the manufacturer’s protocol. The PCR product, which was approximately 1.5 kb in size, was sequenced by capillary sequencing (AIT Biotech, Singapore, Singapore). For identification, DNA sequences were compared with the 16S ribosomal RNA sequences in the Human Oral Microbiome Database. A cutoff of value of 98.5% identity was set (18).
Assessment of pH Tolerance of Bacterial Isolates Bacterial colonies were inoculated into 2 mL buffered BHI broth supplemented with yeast extract, hemin, and vitamin K with pH levels adjusted to 9, 10, and 11 with 10 mol/L potassium hydroxide and incubated anaerobically at 37 C for 3 days. The amount of bacterial growth was measured by optical density at 600 nm. The relationship between optical density and colony-forming units was determined for each species by serial dilution and plating on TSA plate sheep blood agar plates. Based on optical density values, bacterial growth was categorized as none (), weak (+), moderate (++), or heavy (+++).
Results Tests were performed to determine the sustainability of alkaline pH under the culture conditions. As shown in Figure 1, in the absence of bacteria, alkaline pH could be maintained within the range of 9.0 0.2 pH units for a maximum period of 3 days. All 43 samples were culture positive at S1 with amounts ranging from 102–107 colony-forming units as determined by anaerobic culture. A total of 30 samples (70%) were found to harbor alkaline-tolerant bacteria (Table 1). Consistently only 1 alkaline-tolerant bacterial species was isolated from each S1 sample. Among the bacterial isolates obtained, 23 of 30 (77%) were gram-positive bacteria (Table 2), with Pseudoramibacter alactolyticus and Streptococci spp being the most frequently isolated with a prevalence of 54%. The other bacterial isolates obtained were Enterobacter cancerogenus, Fusobacterium nucleatum, and Enterococcus faecalis. Among the 13 culture-positive S2 samples, 8 alkaline-tolerant bacteria were isolated. There was a correlation between the presence of these organisms at S1 and their persistence at S2. Seven of 8 bacterial isolates (88%) retrieved in S2 samples were correspondingly obtained at S1. Streptococcus spp, E. faecalis, E. cancerogenus, and F. nucleatum were among the bacterial isolates obtained at S2. Among the 3 culture-positive samples at S3, all possessed alkaline-tolerant isolates,
Figure 1. pH changes of culture media over time. BHI broth supplemented with yeast extract, hemin, and vitamin K was adjusted to pH 9 and incubated at 37 C anaerobically in the absence of bacteria. pH was taken daily for up to 5 days.
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namely Streptococcus sanguinis and E. faecalis. These species were also isolated from the corresponding S1 and S2 samples. To determine the degree of alkaline tolerance of the bacterial isolates, the growth of these bacteria were further assessed at pH levels of 10 and 11. E. faecalis was the only bacterial species capable of surviving a pH of 11, whereas E. cancerogenus was able to withstand a pH of 10 (Table 3).
Discussion Few studies have addressed the issue of alkaline tolerance among root canal bacteria. In the 2 other studies reported so far, both described the predominance of gram-positive bacteria enduring alkaline conditions, with biofilm forms being more resistant than planktonic cells (14, 19). In this study, we showed that more than two thirds of infected root canal samples harbored alkaline-tolerant bacteria; gram-positive bacteria dominated. The first stage of sampling at S1 began from untreated necrotic pulps to isolate alkaline-tolerant bacteria from the initial colonizing population in the root canal. Thus, isolates at this stage represent bacteria that have not been exposed to alkaline stress but have the inherent ability to grow under laboratory conditions at a pH of 9 even without adaptation from pre-exposure to alkaline medicaments. P. alactolyticus and Streptococcus spp were the most frequently isolated species, whereas E. faecalis occurred only in 2 cases. The differences to the findings of Nakajo et al (14) may be caused by the sampling method adopted in that study in which bacteria from infected root canals were obtained from homogenized dentin powder from extracted teeth. Our results further showed that when alkaline-tolerant bacteria were found in infected root canals, only 1 bacterial isolate was obtained from each canal. The reason behind this observation, although interesting, is currently unclear. This may be attributed to the fact that bacteria surviving in the root canal are present in biofilm communities where alkaline-tolerant microorganisms may produce bacteriocins to suppress the growth of competing bacterial species (20). P. alactolyticus is a strict anaerobic gram-positive rod. Several studies have shown this species to be a common resident of primary root canal infections with a prevalence ranging from 34%–76% (21–23). In the present study, P. alactolyticus was the most frequently isolated bacteria at S1. However, this bacterial species was not obtained from subsequent S2 and S3 samples. Our observations agree with data from a previous study that showed that P. alactolyticus is easily eliminated by endodontic disinfection procedures (24). Our results show that P. alactolyticus, although culturable at a pH of 9, was not able to withstand the higher pH of sodium hypochlorite irrigation and mechanical debridement. This organism may nevertheless play a significant role in the pathogenesis of primary root canal infections. Streptococcus spp was as equally prevalent as P. alactolyticus in S1 samples. Of the 8 isolates obtained at S1, 4 persisted in S2 samples and 2 in S3 samples. Streptococcus sanguinis and Streptococcus oralis appeared to be the most resilient species obtained in this study, showing a high resistance toward instrumentation and antimicrobial irrigants and/or Ca(OH)2 dressing. In a study investigating the diversity of Streptococcus spp in root canals undergoing endodontic therapy, S. gordonii and S. oralis were the most prevalent (25). The presence of gram-positive bacteria, namely Lactobacillus spp, the nonmutans group Streptococci (S. sanguinis, S. gordonii, S. oralis, and Streptococcus mitis), and Enterococcus spp, have been found to be significantly correlated with cases that had undergone chemomechanical procedures and interappointment medicaments (26). These differences may be attributed to variations associated with the patient pool.
Isolation of Alkaline-tolerant Bacteria
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Clinical Research TABLE 1. Alkaline-tolerant Bacterial Species at S1, S2, and S3 Case No
Bacterial species at S1
A43 A45 A49 A50 A54 A61 A63 A65 A67 A71 A79 A90 A94 A97 A100 A116 A124 A127 A143 A144 A146 A148 A150 A154 A157 A158 A159 A162 A179 A181
Bacterial species at S2
Parvimonas micra Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Enterococcus faecalis Enterobacter cancerogenus Fusobacterium nucleatum Pseudoramibacter alactolyticus Klebsiella pneumonia Parvimonas micra Streptococcus oralis Pseudoramibacter alactolyticus Peptostreptococcus stomatis Streptococcus sanguinis Propionibacterium propionicum Streptococcus sanguinis Peptostreptococcus stomatis Fusobacterium nucleatum Streptococcus intermedius Fusobacterium nucleatum Pseudoramibacter alactolyticus Pseudoramibacter alactolyticus Enterobacter cancerogenus Streptococcus sanguinis Streptococcus intermedius Enterobacter cancerogenus Streptococcus sanguinis Streptococcus oralis Enterococcus faecalis
Bacterial species at S3
Enterococcus faecalis Enterobacter cancerogenus
Fusobacterium nucleatum Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus sanguinis
Streptococcus sanguinis Streptococcus oralis Enterococcus faecalis
Enterococcus faecalis
Bacterial counts (cfu/mL) at S1 6 106 2 102 1.6 104 2.2 105 1 105 5.8 107 8 106 5 105 1.3 103 1 107 3.38 107 2.62 107 1.12 105 8 104 7 106 1.82 104 2 106 4.14 106 2.4 102 1.64 106 1.2 105 6.2 104 4 104 2 104 1.6 104 1.02 105 1.74 103 1.2 103 1 104 1.12 107
Case number refers to bacterial species of the respective root canal samples that have been obtained by culturing the samples at a pH of 9. S1, S2, and S3 refer to bacterial species obtained before endodontic treatment, after chemomechanical root canal preparation, and after placement of calcium hydroxide dressing, respectively.
In our sample population, Streptococci and Enterococci spp were found to be the predominant alkaline-tolerant bacterial types that survived chemomechanical treatment and the placement of Ca(OH)2 dressing. These observations may be attributed to the organisms’ ability to overcome stresses associated with root canal treatment such as antimicrobial usage, changes in redox potential, and changes in nutrients (27). As expected, the bacterial load was greatly reduced after chemomechanical treatment. After irrigation and mechanical instrumentation (S2), 8 of 30 samples (27%) with alkaline-tolerant organisms in S1
TABLE 2. Occurrence of Alkaline-tolerant Bacterial Species Bacterial species Gram-positive rod Pseudoramibacter alactolyticus Propionibacterium propionicum Gram-positive cocci Enterococcus faecalis Streptococcus sanguinis Streptococcus intermedius Streptococcus oralis Peptostreptococcus stomatis Parvimonas micra Gram-negative rod Fusobacterium nucleatum Enterobacter cancerogenus Gram-negative cocci Klebsiella pneumonia Total
4
Lew et al.
S1
S2
S3
2 4 2 2 2 2
2 3
1 2
3 3
1 1
1 30
8
8 1
1
3
were recovered. Alkaline-tolerant bacteria isolated at S2 included 4 isolates of Streptococcus spp, 2 isolates of E. faecalis, and 1 isolate each of E. cancerogenus and F. nucleatum. Isolates obtained at the S2 stage represent bacteria that can withstand acute exposure to the high pH of sodium hypochlorite irrigation. The number of culture-positive samples obtained at S3 was 3 of 43 (7%), which is similar to the findings reported by Shuping et al (28). Isolates obtained at the S3 stage represent bacteria that withstood sodium hypochlorite irrigation as well as prolonged exposure to Ca(OH)2 dressing. These bacteria isolates either possess or have acquired robust mechanisms to survive alkaline conditions. Although the numbers obtained were relatively small, it is significant given that all 3 culture-positive samples harbored alkaline-tolerant organisms, namely S. sanguinis and E. faecalis. More importantly, the same organisms were isolated at the respective S1 and S2 stages, suggesting that alkaline-tolerant bacteria belonging to the genus of Streptococcus and Enterococcus may possess an enhanced ability to withstand endodontic treatment procedures. F. nucleatum was the only bacterial isolate obtained at S2 without appearing in the corresponding S1 sample. Possible explanations include culture reversal (6) and contamination from the oral environment during treatment or interappointment interval. Another possible explanation is that the cell count of F. nucleatum was low during the first sampling such that it was not isolatable, but the amounts increased in numbers in the canal to become retrievable after irrigation and instrumentation. Among the alkaline-tolerant bacteria obtained in this study, only E. cancerogenus and E. faecalis were able to persist at a pH $9, enduring a pH of 10 and 11, respectively. It has been reported in numerous studies that E. faecalis is capable of enduring high alkaline conditions JOE — Volume -, Number -, - 2015
Clinical Research TABLE 3. Degree of Alkaline Tolerance of Bacterial Species Bacterial species
pH 9
pH 10
pH 11
Enterococcus faecalis Enterobacter cancerogenus Fusobacterium nucleatum Pseudoramibacter alactolyticus Klebsiella pneumonia Streptococcus oralis Parvimonas micra Peptostreptococcus stomatis Propionibacterium propionicum Fusobacterium nucleatum Enterobacter cancerogenus Streptococcus sanguinis Streptococcus intermedius Peptostreptococcus stomatis Fusobacterium nucleatum
+++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
++ +
+
, no growth; +, weak growth; ++, moderate growth; +++, heavy growth.
up to a pH of 11 (29–31), likely through the action of proton pumps, which allow the bacteria to maintain intracellular pH homeostasis (32). The cellular mechanism behind E. cancerogenus’ ability to withstand a pH of up to 10 is currently unknown and awaits future studies. The survival of alkaline-tolerant bacteria at S2 and S3 in the present study may be attributed to the influence of dentin on the antimicrobial actions of sodium hypochlorite and Ca(OH)2. The time required for sodium hypochlorite to eradicate bacteria increased significantly if it had been preincubated with dentin powder (33). In a recent study, Macedo et al (34) further reported that the pH of unbuffered sodium hypochlorite solution dropped from a pH of 12 to a pH of 10.5 after 50 seconds of contact time with dentin. The antibacterial effects of Ca(OH)2 have been controversial because of inconsistencies in obtaining negative cultures from canals treated with Ca(OH)2 (6, 7, 8, 12, 29). Although this may be partially attributed to a lack of direct contact of microorganisms with Ca(OH)2 in the root canal, increasing evidence show that dentin is able to buffer the high pH of Ca(OH)2, which is essential for its antimicrobial action (33, 35). One of the limitations of this study was that not all bacteria obtained from sampling are isolatable. Although we selected a media that allowed the growth of as wide a range of bacteria as possible, bacteria with more stringent or unique culture requirements than those in this study would not be isolated. Alkaline pH was also not sustainable for more than 3 days under anaerobic conditions. The cultures were isolated no later than 3 days to avoid growth of other bacteria when the pH drops below 9. Thus, slower-growing potential alkaline bacteria would not be isolated. Another limitation of this study was the sampling method in which paper points were unable to reach the irregularities of the root canal system such as the fins, isthmuses, lateral canals, apical delta, and dentinal tubules. Hence, the alkaline-resistant bacteria isolated in this study were likely representative of the microflora in the main root canals. The current study was undertaken to characterize the relationship between the tolerance of bacterial isolates to a pH of 9 and survival. It remains to be determined if these bacterial isolates adopt universal physiological responses to endure high pH conditions. Such knowledge may allow us to develop enhanced antimicrobial approaches against these microorganisms.
Conclusions This study describes alkaline-tolerant species present at various stages of endodontic therapy. Although the mechanisms adopted by these alkaline-tolerant bacterial isolates remain to be JOE — Volume -, Number -, - 2015
determined, our results suggested that the presence of these bacteria in primary root canal infections could pose a challenge to current treatment efficacy.
Acknowledgments Supported by a grant (R221-000-051-112) from the Ministry of Education Singapore. The authors deny any conflicts of interests related to this study.
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JOE — Volume -, Number -, - 2015
