Clinical Research
Short-chain Fatty Acids in Infected Root Canals of Teeth with Apical Periodontitis before and after Treatment Jose Claudio Provenzano, PhD,* Isabela N. R^ o¸cas, PhD, Luıs Fernando D. Tavares, PhD,† † Bianca Cruz Neves, PhD, and Jose F. Siqueira, Jr, PhD* Abstract Introduction: Short-chain fatty acids (SCFAs) are bacterial metabolic end products that may function as virulence factors. This study evaluated the occurrence of SCFAs in infected root canals before and after treatment. Methods: Samples were taken from root canals of teeth with apical periodontitis before (S1) and after (S2) chemomechanical preparation with either NaOCl or chlorhexidine as the irrigant and then after interappointment medication with calcium hydroxide (S3). High-performance liquid chromatography was used for detection of SCFAs. Selected bacterial taxa that are recognized producers of the target SCFAs were identified by real-time polymerase chain reaction. Results: Butyric acid was the most common fatty acid in S1, followed by propionic acid. Both molecules were also found in S2 and S3 from both NaOCl and chlorhexidine groups. Lactic acid was not present in detectable levels in S1, but it occurred in 1 postinstrumentation sample and in 9 samples taken after calcium hydroxide medication. Of the target taxa, Fusobacterium nucleatum was the most prevalent in S1 (76%), followed by members of the Actinobacteria phylum (71%), Streptococcus species (59%), and Parvimonas micra (53%). Gram-positive taxa, especially streptococci, were the most prevalent bacteria in S2 and S3. SCFA detection was matched with the respective potential producer species in most cases. Conclusions: This first report of SCFAs in infected root canals suggests that these molecules may play a role in the pathogenesis of apical periodontitis. Significance of persistence of SCFAs after treatment and its effects on the long-term outcome await elucidation. (J Endod 2015;-:1–5)
Key Words Apical periodontitis, butyric acid, endodontic treatment, lactic acid, propionic acid, root canal infection, shortchain fatty acids
B
acteria infecting the root canals of teeth with apical periodontitis are usually organized in mixed biofilm communities (1). The pathogenic ability of multispecies biofilms is certainly related to the accumulation of virulence factors and antigens from the different component species. This bacterial ‘‘soup’’ may contact the periradicular tissues via apical foramen/foramina and evoke inflammation, with consequent formation of apical periodontitis (2). Virulence factors that are expected to concentrate in the biofilm include bacterial cellular constituents (eg, lipopolysaccharide, peptidoglycan, lipoteichoic acid, complex lipids, etc), and metabolic end products (3, 4). Several end products of the bacterial metabolism are released to the extracellular environment and may be toxic to host cells, cause degradation of constituents of the extracellular matrix of the connective tissue, and interfere with host defense processes (5–7). Among them, short-chain fatty acids (SCFAs) are low-molecular-weight molecules that have been regarded as potential virulence factors involved in the pathogenesis of inflammatory conditions such as periodontal diseases (8, 9). Gingival inflammation has been directly and significantly correlated with SCFA concentrations in the gingival crevice (10). In addition, propionic and butyric acid concentrations in the crevicular fluid have been significantly associated with clinical measures of periodontal disease severity and inflammation and with the total microbial load (11). Concentrations of lactic, propionic, butyric, and isovaleric acids have been shown to decrease significantly in the gingival crevicular fluid of patients with marginal periodontitis after treatment, reaching levels comparable with the healthy control group (12). A role for SCFA in the pathogenesis of apical periodontitis, which is also an inflammatory disease of bacterial etiology, has been suggested (3). (CH2)x COOH; X < 3 thus C # 5] include volatile acids SCFAs [CH3 (eg, propionic, butyric, acetic, and isovaleric) and non-volatile acids (eg, lactic and succinic acids) (13). Because endodontic pathogens are known to produce many SCFAs in vitro, these putative virulence factors are expected to accumulate in infected root canals. Nevertheless, no study has so far directly screened endodontic clinical samples for the presence of these bacterial metabolic end products. Detection of SCFAs in infected root canals is important in several aspects. First, it contributes to the knowledge of virulence factors potentially involved in the pathogenesis of apical periodontitis. Second, occurrence of these metabolic factors in pretreatment and post-treatment samples may permit inferences to be made as to how bacteria react to treatment procedures. Finally, because there is high interindividual variability in the bacterial diversity of endodontic infections and no specific species has been consistently associated with clinical symptoms or treatment outcome (14), there is a trend to look for bacterial products that may serve as biomarkers for certain clinical conditions (15, 16). This is because some redundancy in distinct communities is expected in terms of bacterial
From the *Molecular Microbiology Laboratory, Department of Endodontics, Faculty of Dentistry, Estacio de Sa University, Rio de Janeiro, Rio de Janeiro, Brazil; and Laboratory of Molecular Microbiology and Proteins, Department of Biochemistry, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil. Address requests for reprints to Dr Jose Claudio Provenzano, Faculty of Dentistry, Estacio de Sa University, Av Alfredo Baltazar da Silveira, 580/cobertura, Recreio, Rio de Janeiro, RJ, Brazil 22790-710. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.02.006 †
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Short-chain Fatty Acids in Infected Canals
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Clinical Research physiology and function, and consequently, the variability in bacterial products released in the environment is conceivably lower than the species composition. This is typical when one considers that several different species can produce the very same enzyme or metabolic product. This study was undertaken to evaluate the presence and prevalence of several SCFAs in infected root canals before and after treatment with either sodium hypochlorite (NaOCl) or chlorhexidine (CHX) as the irrigant and interappointment dressing with a calcium hydroxide paste. Identification of selected bacterial taxa that are potential producers of the SCFAs under investigation was also performed.
Materials and Methods Subjects and Sample Taking Samples were taken from patients who had been referred for root canal treatment to the Department of Endodontics, Estacio de Sa University. Only single-rooted and single-canalled teeth from 18 adult patients (ages ranging from 20 to 39 years) with carious lesions, necrotic pulps confirmed by pulp tests, and clinical and radiographic evidence of asymptomatic apical periodontitis were included in this study. Exclusion criteria included teeth with gross carious lesions, teeth with root or crown fracture, teeth subjected to previous endodontic treatment, patients who received antibiotic therapy within the previous 3 months, symptomatic teeth, and patients with periodontal pockets deeper than 4 mm. The study was approved by the Ethics Committee at Estacio de Sa University, Rio de Janeiro, Brazil, and written informed consent was obtained from all the patients. Samples were taken from the root canals as follows. After the tooth crown was cleansed with pumice, a rubber dam was placed, and the tooth and the surrounding field were decontaminated by a protocol that used 3% hydrogen peroxide followed by 2.5% NaOCl solution (17). Complete access preparations were made by using sterile burs without water spray. The operative field, including the pulp chamber, was again swabbed with 2.5% NaOCl, which was then inactivated with sterile 5% sodium thiosulfate. Sterility control samples were taken from the tooth surface with sterile paper points. All the teeth included in the study had sterility control samples uniformly negative after polymerase chain reaction (PCR) with universal bacterial primers. The root canal was filled with 10 mmol/L Tris-HCl (pH 8.0), and a K-type file no. 15 was introduced up to approximately 1 mm short of the root apex, on the basis of radiographs, and used to gently file the canal walls. Afterwards, the fluid in the canal was aspirated by using a sterile disposable syringe and transferred to a cryotube. These sampling procedures were repeated several times until 100 mL sampled fluid was obtained. Sample was immediately frozen at 80 C. This initial root canal sample was called S1. Each root canal was instrumented at the same visit by using BioRaCe instruments (FKG Dentaire, La Chaux-de-Fonds, Switzerland), with the working length established 1 mm short of the radiographic apex. Master apical files ranged from BR5 (40/.04) to BR7 (60/.04), depending on both the root anatomy and the preoperative root canal diameter. Patency of the apical foramen was confirmed with #20 Ktype file throughout the procedures. Irrigation was performed with either 2.5% NaOCl (9 teeth) or 2% CHX (FGM, Joinvile, SC, Brazil) (9 teeth) by using disposable syringes and NaviTip needles (Ultradent, South Jordan, UT) inserted up to 4 mm short of the working length. Two milliliters of the irrigating solution (NaOCl or CHX) was used after each file size. After preparation, the root canal was dried and then flushed with 5 mL either 10% sodium thiosulfate solution or mixture of 0.07% lecithin, 0.5% Tween 80, and 5% sodium thiosulfate to neutralize 2
Provenzano et al.
any residual NaOCl or CHX, respectively. Postinstrumentation (S2) samples were taken from the root canals as described for S1 samples. Smear layer was removed by using 17% EDTA for 3 minutes. The canal was dried with paper points, medicated with Ca(OH)2 paste in camphorated paramonochlorophenol and glycerin, and placed in the canals by means of lentulo spiral fillers. A radiograph was taken to ensure proper placement of the paste in the canal, and the access cavity was filled with at least 4-mm thickness of temporary cement (Coltosol; Coltene/Whaledent Inc, Cuyahoga Falls, OH). The second appointment was scheduled 1 week later. At this time, the tooth was isolated with a rubber dam, the operative field was disinfected before and after removal of the temporary cement, NaOCl was neutralized, and a sterility control sample was taken. All these procedures were carried out as outlined previously. The Ca(OH)2 paste was rinsed out of the canal by using sterile saline solution and the master apical file. The root canal walls were gently filed, and a postmedication sample (S3) was taken from the canal as described for S1. The canal was filled with gutta-percha and Sealer 26 (Dentsply, Petropolis, RJ, Brazil) by using cold lateral compaction, the tooth was temporized with glass ionomer cement, and a permanent restoration was planned. All clinical procedures were conducted by one experienced endodontist (J.C.P.).
High-performance Liquid Chromatography Analysis Detection of SCFAs was performed directly in clinical samples. Fifty-microliter aliquots of each sample were diluted in 5 mmol/L H2SO4 at 1:2 ratio, followed by centrifugation at 10,000 rpm/10 min. Dilution of clinical samples was needed to reduce possible interferences related to the injection of complex samples in high-performance liquid chromatography (HPLC) and to keep samples at the detection limit, because they were highly concentrated and generated very intense peaks. Analysis were conducted in HPLC equipped with DGU-20A5 degasser, LC-20AT liquid chromatograph, SIL-20A auto sampler, CTO-20A column oven, RID-10A refractive index detector, SPD-M20 A diode array detector, FRC-10A fraction collector, and CBM-20A communications bus module (Shimadzu, Kyoto, Japan). Aminex HPX-87H column (300 7, 8 mm; Bio-Rad Laboratories Ltd, Hercules, CA) was used with a mobile phase composed of 5 mmol/L H2SO4 and a flow rate of 0.6 mL/min. Total run time was 30 minutes, and the RID was used for the analysis. The column oven was set to 50 C. Commercial standards of the analyzed fatty acids (Sigma-Aldrich, S~ao Paulo, SP, Brazil) were injected before the sample analysis. A total of 20 mL of each sample and standards was injected in the HPLC. Negative controls consisted of HPLC ultrapure water. Retention time and detection limits for each fatty acid targeted are depicted in Table 1. Experiments were run in duplicate. Real-time PCR Analysis DNA was extracted from a volume of 50 mL of each clinical sample by using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA), following the protocol recommended by the manufacturer. DNA extracts served as templates in a presence/absence real-time PCR assay for the detection of selected endodontic pathogens that are potential producers of the SCFAs targeted. The 16S rRNA gene-based PCR primers were specific for Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, members of the Dialister and Streptococcus genera, and the Actinobacteria phylum. Universal bacterial 16S rRNA gene-based primers were also used to serve as controls. PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on an ABI 7500 real-time JOE — Volume -, Number -, - 2015
Clinical Research samples, but it occurred in 1 S2 sample (CHX group) and 9 S3 samples (5 from the NaOCl group and 4 from the CHX group). Acetic acid was found in 1 post-treatment sample. Data are shown in Table 2.
TABLE 1. HPLC Data of the Analyzed Fatty Acids Fatty acid
Molecular formula
Retention time (min)
Detection limit (mmol/L)
Acetic acid Butyric acid Glycolic acid Isovaleric acid Lactic acid Maleic acid Propionic acid
C2H4O2 C4H8O2 C2H4O3 C5H10O2 C3H6O3 C4H4O4 C3H6O2
14.523 21.372 12.256 24.607 11.839 17.036 9.429
0.014 0.63 0.55 0.058 0.11 0.33 1.49
PCR instrument (Applied Biosystems) in a total reaction volume of 20 mL. Primers in a concentration of 1 mmol/L each and DNA extract volume of 2 mL were added to the PCR master mix in MicroAmp Optical 96well reaction plates. Plates were sealed, centrifuged, and subjected to amplification. Primer sequences and cycling conditions were as described and validated previously (18–23). Negative and positive controls were included in each batch of samples analyzed. Negative samples consisted of sterile ultrapure water instead of the sample. Positive controls consisted of DNA extracted from pure cultures of representative strains of the target species or groups, including F. nucleatum (ATCC 25586 and ATCC 10953), P. micra (ATCC 33270), P. endodontalis (ATCC 35406), P. alactolyticus (C11b-d), Streptococcus mutans (ATCC 25175), and Actinomyces species and Dialister invisus isolated in a previous study (24). Experiments were run in triplicate for all samples and controls, and specificity of the amplification was always determined with the use of melting curve analysis.
Data Analysis Prevalence of the fatty acids and target species was recorded as the percentage of cases examined. Attempts were made to correlate the SCFA found in the sample concomitantly with its potential producer species.
Real-time PCR Analysis Real-time PCR analysis by using universal 16S rRNA gene-based primers revealed that all S1 samples were positive for total bacteria. Overall, 12 of 17 S2 samples (71%) and 8 of 17 S3 samples (47%) still had detectable bacterial levels. In NaOCl group, 6 of 9 S2 samples (67%) and 4 of 9 S3 samples (44%) exhibited positive PCR results for bacteria, whereas respective figures for the CHX group were 6 of 8 S2 samples (75%) and 4 of 8 S3 samples (50%). All the target taxa were found in S1 samples (Table 3). The most prevalent were F. nucleatum (76%), members of the Actinobacteria phylum (71%), Streptococcus species (59%), and P. micra (53%). The target taxa were also found in at least 1 post-treatment sample. Gram-positive taxa were much more prevalent in S2 and S3 than the gram-negative taxa, with Streptococcus species as the most prevalent bacteria regardless of the irrigant used. Matching each SCFA with its potential producers in the same clinical sample demonstrated that of the 26 samples positive for butyric acid, F. nucleatum was found in 14, P. alactolyticus in 8, and P. endodontalis in 5. None of the target taxa that are known butyric acid producers were found in 8 samples positive for this fatty acid. Of the 12 samples positive for propionic acid, F. nucleatum and members of the Actinobacteria phylum were detected in 8 and P. endodontalis and Dialister species in 3 samples each. None of the potential propionic acid producers were found in 2 samples positive for this acid. Lactic acid was found in 10 samples, 3 of which had Actinobacteria, and 2 had P. micra and Streptococcus species. Five lactic acid–positive samples yielded none of the potential producers. Succinic acid was found in 2 samples, one presenting P. micra and the other Actinobacteria. In the sample positive for acetic acid, 3 potential producers were found: Actinobacteria, Streptococcus, and P. micra.
Results
Discussion
SFCA Analysis One sample from the CHX group was lost during preparation for HPLC analysis, and the case was excluded. HPLC screening revealed that butyric acid was the most common fatty acid in S1 samples (10 of 17 samples, 59%). It was also frequently found in S2 and S3 samples from both NaOCl and CHX groups. Propionic acid was detected in 7 S1 samples (41%) and also in post-treatment samples from both NaOCl and CHX groups. Of the other fatty acids targeted, only succinic acid was also found in S1. Lactic acid was not present in detectable levels in S1
SCFA are well-known metabolic end products of anaerobic bacteria. Several candidate endodontic pathogens produce SCFAs, and because they are usually arranged in biofilms, these substances are likely to accumulate in the canal. To the best of our knowledge, this is the first study to evaluate the occurrence of SCFAs directly in endodontic clinical samples before and after treatment. Butyric and propionic acids were frequently found in initial samples. This was somewhat expected because many of the candidate endodontic pathogens present in primary infections are anaerobic bacteria
TABLE 2. SFCAs in Infected Root Canals before (S1) and after Root Canal Preparation (S2) with Either NaOCl or CHX as the Irrigant and after Intracanal Medication with Calcium Hydroxide (S3) NaOCl (n = 9)
CHX (n = 8)
Total (n = 17)
Fatty acids
S1
S2
S3
S1
S2
S3
S1
S2
S3
Butyric acid Propionic acid Lactic acid Succinic acid Acetic acid Maleic acid Glycolic acid Isovaleric acid
5 (56)* 5 (56) ND ND ND ND ND ND
4 (44) 2 (22) ND 1 (11) 1 (11) ND ND ND
4 (44) 1 (11) 5 (56) ND ND ND ND ND
5 (63) 2 (25) ND 1 (13) ND ND ND ND
4 (50) ND 1 (13) ND ND ND ND ND
4 (50) 2 (25) 4 (50) ND ND ND ND ND
10 (59) 7 (41) ND 1 (6) ND ND ND ND
8 (47) 2 (12) 1 (6) 1 (6) 1 (6) ND ND ND
8 (47) 3 (18) 9 (53) ND ND ND ND ND
ND, not detected. *Number of positive cases (percentage).
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Short-chain Fatty Acids in Infected Canals
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Clinical Research TABLE 3. Selected Endodontic Pathogens in Infected Root Canals before (S1) and after Root Canal Preparation (S2) with Either NaOCl or CHX as the Irrigant and after Intracanal Medication with Calcium Hydroxide (S3) NaOCl (n = 9)
CHX (n = 8)
Total (n = 17)
Bacteria
S1
S2
S3
S1
S2
S3
S1
S2
S3
Total bacteria Streptococcus species Actinobacteria P. micra F. nucleatum P. alactolyticus P. endodontalis Dialister species
9 (100)* 4 (44) 4 (44) 8 (89) 6 (67) 1 (11) 5 (56) 4 (44)
6 (67) 6 (67) 3 (33) 5 (56) 2 (22) 1 (11) 1 (11) 0 (0)
4 (44) 3 (33) 2 (22) 4 (44) 1 (11) 1 (11) 1 (11) 0 (0)
8 (100) 6 (75) 8 (100) 1 (13) 7 (88) 7 (88) 3 (38) 1 (13)
6 (75) 5 (63) 5 (63) 0 (0) 3 (38) 3 (38) 0 (0) 1 (13)
4 (50) 3 (38) 4 (50) 0 (0) 1 (13) 3 (38) 0 (0) 0 (0)
17 (100) 10 (59) 12 (71) 9 (53) 13 (76) 8 (47) 8 (47) 5 (29)
12 (71) 11 (65) 8 (47) 5 (29) 5 (29) 4 (24) 1 (6) 1 (6)
8 (47) 6 (35) 6 (35) 4 (24) 2 (12) 4 (24) 1 (6) 0 (0)
*Number of positive cases (percentage).
known to produce these fatty acids in vitro. Both substances were also found in some samples taken after preparation by using either NaOCl or CHX as the irrigant. This indicates either that these SCFAs were not completely eliminated from the canals by preparation or that bacteria remaining in these canals were still metabolically active. Other SCFAs detected in this study included succinic and acetic acids, both in lower prevalences, and lactic acid. The main biological effects of SCFAs related to inflammation include toxicity to several cell types and effects on neutrophil chemotaxis, phagocytosis, and cytokine production. Butyric and propionic acids have been demonstrated to be cytotoxic to Vero cells (5, 25), which are a line of monkey cells used for many purposes including screening for toxicity of bacteria to mammalian cells. Butyric acid can inhibit T-cell proliferation (7) and induce apoptosis in monocytes, T and B lymphocytes, and epithelial cells (26–30). Butyric acid also induces production of reactive oxygen species and the impairment of cell growth in fibroblasts (31). Effects of SCFAs on chemotaxis and phagocytosis have also been reported. Butyric, succinic, and propionic acids stimulate neutrophil migration to inflamed sites and suppress the phagocytic capacity of phagocytes (7, 32, 33). Succinic acid at concentrations commonly found in clinical abscesses has been shown to impair the antimicrobial activity of neutrophils (34, 35). Butyric acid can stimulate the production of proinflammatory cytokines by monocytes and neutrophils (7, 30). All these biological activities and the detection of these SCFAs in samples from primary endodontic infections lend credence to the assertion that these molecules can play a causative role (along with other virulence factors) in apical periodontitis. All the bacterial taxa targeted in the real-time PCR assay have been frequently found in primary endodontic infections (36–39), which was confirmed in this study. Streptococci and Actinobacteria were more commonly detected in post-treatment samples. This is also in agreement with previous studies (20, 40–42). Selection of the target taxa was based on the fact that they are very prevalent in primary infections and are recognized in vitro producers of the SCFAs under investigation. For instance, butyric acid is a by-product of P. endodontalis, F. nucleatum, and P. alactolyticus, whereas propionic acid is produced by the former 2 species and members of the Dialister genus and Actinobacteria phylum. P. micra and Actinobacteria produce succinic acid. All the target species can generate acetic acid. In most cases, there was a correlation between the fatty acid detected and the presence of a target species that is a potential producer of that fatty acid. However, matching the acid to its producer did not occur in some cases. This is because endodontic infections are polymicrobial infections, and it would be virtually impossible to target all the potential producer species. Further studies that use broad-range microbial identification are required for a more profound correlative analysis.
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Particularly noteworthy was the high incidence of lactic acid in samples taken after intracanal medication with calcium hydroxide. Lactic acid is the most commonly recognized SCFA because of its occurrence at the termination of the anaerobic glycolytic cascade. In the oral environment, lactic acid is widely known because of its generation by oral bacteria in association with caries (43). Although this fatty acid was not detected in initial samples and was found in only 1 postinstrumentation sample, it was present in 9 post-medication samples. This may suggest a possible mechanism by which some bacteria respond to the alkaline stress induced by calcium hydroxide. Alternatively, the possibility also exists that lactic acid production is merely a characteristic of bacteria surviving the effects of calcium hydroxide. Potential lactic acid producers, such as Streptococcus species, Actinobacteria, and P. micra, were detected in several samples positive for this fatty acid. Other lactic acid producers not targeted in this study might well be present in these samples. This is especially applicable to the 5 lactic acid– positive samples in which none of the target species were found. Further studies should elaborate more on this increased detection of lactic acid after calcium hydroxide challenge and the producer species. In conclusion, this first report of the occurrence of SCFAs in infected root canals suggests that these bacterial products may play a role as virulence factors in the pathogenesis of apical periodontitis. These bacterial molecules were also found in post-treatment samples, always in association with bacterial presence. Significance of persistence of SCFAs after treatment and its effects on long-term outcome remain to be determined. Also, further quantitative studies are required to determine association of these substances with symptoms and ability of treatment to reduce their concentrations. The high occurrence of lactic acid after calcium hydroxide medication also deserves further investigations.
Acknowledgments This study was supported by grants from Fundac¸~ao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), Brazilian governmental institutions. The authors deny any conflicts of interest related to this study.
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Clinical Research 5. Grenier D, Mayrand D. Cytotoxic effects of culture supernatants of oral bacteria and various organic acids on Vero cells. Can J Microbiol 1985;31:302–4. 6. van Steenbergen TJM, van der Mispel LMS, de Graaff J. Effect of ammonia and volatile fatty acids produced by oral bacteria on tissue culture cells. J Dent Res 1986; 65:909–12. 7. Eftimiadi C, Stashenko P, Tonetti M, et al. Divergent effect of the anaerobic bacteria by-product butyric acid on the immune response: suppression of T-lymphocyte proliferation and stimulation of interleukin-1 beta production. Oral Microbiol Immunol 1991;6:17–23. 8. Miragliotta G, Mosca A, Del Prete R. The production of short-chain fatty acids by periodontopathic bacteria contributes to the impairment of local host defence. Med Hypotheses 1993;41:462–4. 9. Tonetti M, Eftimiadi C, Damiani G, et al. Short chain fatty acids present in periodontal pockets may play a role in human periodontal diseases. J Periodontal Res 1987;22:190–1. 10. Niederman R, Buyle-Bodin Y, Lu BY, et al. The relationship of gingival crevicular fluid short chain carboxylic acid concentration to gingival inflammation. J Clin Periodontol 1996;23:743–9. 11. Niederman R, Buyle-Bodin Y, Lu BY, et al. Short-chain carboxylic acid concentration in human gingival crevicular fluid. J Dent Res 1997;76:575–9. 12. Qiqiang L, Huanxin M, Xuejun G. Longitudinal study of volatile fatty acids in the gingival crevicular fluid of patients with periodontitis before and after nonsurgical therapy. J Periodontal Res 2012;47:740–9. 13. Niederman R, Zhang J, Kashket S. Short-chain carboxylic-acid-stimulated, PMNmediated gingival inflammation. Crit Rev Oral Biol Med 1997;8:269–90. 14. Siqueira JF, R^oc¸as IN. Present status and future directions in endodontic microbiology. Endod Topics 2014;30:3–22. 15. Provenzano JC, Siqueira JF Jr, R^oc¸as IN, et al. Metaproteome analysis of endodontic infections in association with different clinical conditions. PLoS One 2013;8:e76108. 16. Nandakumar R, Madayiputhiya N, Fouad AF. Proteomic analysis of endodontic infections by liquid chromatography-tandem mass spectrometry. Oral Microbiol Immunol 2009;24:347–52. 17. Siqueira JF Jr, R^oc¸as IN, Souto R, et al. Checkerboard DNA-DNA hybridization analysis of endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;89:744–8. 18. Siqueira JF Jr, R^oc¸as IN. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:85–94. 19. R^oc¸as IN, Siqueira JF Jr. Characterization of Dialister species in infected root canals. J Endod 2006;32:1057–61. 20. Neves MA, R^oc¸as IN, Siqueira JF Jr. Clinical antibacterial effectiveness of the selfadjusting file system. Int Endod J 2014;47:356–65. 21. Philippot L, Tscherko D, Bru D, Kandeler E. Distribution of high bacterial taxa across the chronosequence of two alpine glacier forelands. Microb Ecol 2011; 61:303–12. 22. Rudney JD, Pan Y, Chen R. Streptococcal diversity in oral biofilms with respect to salivary function. Arch Oral Biol 2003;48:475–93. 23. Dalwai F, Spratt DA, Pratten J. Use of quantitative PCR and culture methods to characterize ecological flux in bacterial biofilms. J Clin Microbiol 2007;45:3072–6.
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24. Siqueira JF Jr, R^oc¸as IN, Paiva SSM, et al. Cultivable bacteria in infected root canals as identified by 16S rRNA gene sequencing. Oral Microbiol Immunol 2007;22: 266–71. 25. Touw JJ, van Steenbergen TJ, De Graaff J. Butyrate: a cytotoxin for Vero cells produced by Bacteroides gingivalis and Bacteroides asaccharolyticus. Antonie Van Leeuwenhoek 1982;48:315–25. 26. Abe K. Butyric acid induces apoptosis in both human monocytes and lymphocytes equivalently. J Oral Sci 2012;54:7–14. 27. Kurita-Ochiai T, Amano S, Fukushima K, Ochiai K. Cellular events involved in butyric acid-induced T cell apoptosis. J Immunol 2003;171:3576–84. 28. Kurita-Ochiai T, Ochiai K, Fukushima K. Butyric-acid-induced apoptosis in murine thymocytes and splenic T- and B-cells occurs in the absence of p53. J Dent Res 2000;79:1948–54. 29. Kurita-Ochiai T, Fukushima K, Ochiai K. Butyric acid-induced apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Infect Immun 1997;65: 35–41. 30. Tsuda H, Ochiai K, Suzuki N, Otsuka K. Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells. J Periodontal Res 2010;45:626–34. 31. Chang MC, Tsai YL, Chen YW, et al. Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts. J Periodontal Res 2013;48:66–73. 32. Vinolo MA, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by short chain fatty acids. Nutrients 2011;3:858–76. 33. Vinolo MA, Rodrigues HG, Hatanaka E, et al. Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites. Clin Sci (Lond) 2009;117:331–8. 34. Rotstein OD, Pruett TL, Fiegel VD, et al. Succinic acid: a metabolic by-product of Bacteroides species inhibits polymorphonuclear leukocyte function. Infect Immun 1985;48:402–8. 35. Rotstein OD. Interactions between leukocytes and anaerobic bacteria in polymicrobial surgical infections. Clin Infect Dis 1993;16(Suppl 4)):S190–4. 36. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257–62. 37. Gomes BP, Pinheiro ET, Gade-Neto CR, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71–6. 38. R^oc¸as IN, Siqueira JF Jr. Root canal microbiota of teeth with chronic apical periodontitis. J Clin Microbiol 2008;46:3599–606. 39. Sakamoto M, R^oc¸as IN, Siqueira JF Jr, Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol 2006;21:112–22. 40. Chavez de Paz L, 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;100:232–41. 41. Chavez de Paz LE, Molander A, Dahlen G. Gram-positive rods prevailing in teeth with apical periodontitis undergoing root canal treatment. Int Endod J 2004;37:579–87. 42. Sakamoto M, Siqueira JF Jr, R^oc¸as IN, Benno Y. Bacterial reduction and persistence after endodontic treatment procedures. Oral Microbiol Immunol 2007;22:19–23. 43. Margolis HC, Moreno EC. Composition and cariogenic potential of dental plaque fluid. Crit Rev Oral Biol Med 1994;5:1–25.
Short-chain Fatty Acids in Infected Canals
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Short-chain Fatty Acids in Infected Root Canals of Teeth with Apical Periodontitis before and after Treatment Jose Claudio Provenzano, PhD,* Isabela N. R^ o¸cas, PhD, Luıs Fernando D. Tavares, PhD,† † Bianca Cruz Neves, PhD, and Jose F. Siqueira, Jr, PhD* Abstract Introduction: Short-chain fatty acids (SCFAs) are bacterial metabolic end products that may function as virulence factors. This study evaluated the occurrence of SCFAs in infected root canals before and after treatment. Methods: Samples were taken from root canals of teeth with apical periodontitis before (S1) and after (S2) chemomechanical preparation with either NaOCl or chlorhexidine as the irrigant and then after interappointment medication with calcium hydroxide (S3). High-performance liquid chromatography was used for detection of SCFAs. Selected bacterial taxa that are recognized producers of the target SCFAs were identified by real-time polymerase chain reaction. Results: Butyric acid was the most common fatty acid in S1, followed by propionic acid. Both molecules were also found in S2 and S3 from both NaOCl and chlorhexidine groups. Lactic acid was not present in detectable levels in S1, but it occurred in 1 postinstrumentation sample and in 9 samples taken after calcium hydroxide medication. Of the target taxa, Fusobacterium nucleatum was the most prevalent in S1 (76%), followed by members of the Actinobacteria phylum (71%), Streptococcus species (59%), and Parvimonas micra (53%). Gram-positive taxa, especially streptococci, were the most prevalent bacteria in S2 and S3. SCFA detection was matched with the respective potential producer species in most cases. Conclusions: This first report of SCFAs in infected root canals suggests that these molecules may play a role in the pathogenesis of apical periodontitis. Significance of persistence of SCFAs after treatment and its effects on the long-term outcome await elucidation. (J Endod 2015;-:1–5)
Key Words Apical periodontitis, butyric acid, endodontic treatment, lactic acid, propionic acid, root canal infection, shortchain fatty acids
B
acteria infecting the root canals of teeth with apical periodontitis are usually organized in mixed biofilm communities (1). The pathogenic ability of multispecies biofilms is certainly related to the accumulation of virulence factors and antigens from the different component species. This bacterial ‘‘soup’’ may contact the periradicular tissues via apical foramen/foramina and evoke inflammation, with consequent formation of apical periodontitis (2). Virulence factors that are expected to concentrate in the biofilm include bacterial cellular constituents (eg, lipopolysaccharide, peptidoglycan, lipoteichoic acid, complex lipids, etc), and metabolic end products (3, 4). Several end products of the bacterial metabolism are released to the extracellular environment and may be toxic to host cells, cause degradation of constituents of the extracellular matrix of the connective tissue, and interfere with host defense processes (5–7). Among them, short-chain fatty acids (SCFAs) are low-molecular-weight molecules that have been regarded as potential virulence factors involved in the pathogenesis of inflammatory conditions such as periodontal diseases (8, 9). Gingival inflammation has been directly and significantly correlated with SCFA concentrations in the gingival crevice (10). In addition, propionic and butyric acid concentrations in the crevicular fluid have been significantly associated with clinical measures of periodontal disease severity and inflammation and with the total microbial load (11). Concentrations of lactic, propionic, butyric, and isovaleric acids have been shown to decrease significantly in the gingival crevicular fluid of patients with marginal periodontitis after treatment, reaching levels comparable with the healthy control group (12). A role for SCFA in the pathogenesis of apical periodontitis, which is also an inflammatory disease of bacterial etiology, has been suggested (3). (CH2)x COOH; X < 3 thus C # 5] include volatile acids SCFAs [CH3 (eg, propionic, butyric, acetic, and isovaleric) and non-volatile acids (eg, lactic and succinic acids) (13). Because endodontic pathogens are known to produce many SCFAs in vitro, these putative virulence factors are expected to accumulate in infected root canals. Nevertheless, no study has so far directly screened endodontic clinical samples for the presence of these bacterial metabolic end products. Detection of SCFAs in infected root canals is important in several aspects. First, it contributes to the knowledge of virulence factors potentially involved in the pathogenesis of apical periodontitis. Second, occurrence of these metabolic factors in pretreatment and post-treatment samples may permit inferences to be made as to how bacteria react to treatment procedures. Finally, because there is high interindividual variability in the bacterial diversity of endodontic infections and no specific species has been consistently associated with clinical symptoms or treatment outcome (14), there is a trend to look for bacterial products that may serve as biomarkers for certain clinical conditions (15, 16). This is because some redundancy in distinct communities is expected in terms of bacterial
From the *Molecular Microbiology Laboratory, Department of Endodontics, Faculty of Dentistry, Estacio de Sa University, Rio de Janeiro, Rio de Janeiro, Brazil; and Laboratory of Molecular Microbiology and Proteins, Department of Biochemistry, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil. Address requests for reprints to Dr Jose Claudio Provenzano, Faculty of Dentistry, Estacio de Sa University, Av Alfredo Baltazar da Silveira, 580/cobertura, Recreio, Rio de Janeiro, RJ, Brazil 22790-710. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.02.006 †
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Short-chain Fatty Acids in Infected Canals
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Clinical Research physiology and function, and consequently, the variability in bacterial products released in the environment is conceivably lower than the species composition. This is typical when one considers that several different species can produce the very same enzyme or metabolic product. This study was undertaken to evaluate the presence and prevalence of several SCFAs in infected root canals before and after treatment with either sodium hypochlorite (NaOCl) or chlorhexidine (CHX) as the irrigant and interappointment dressing with a calcium hydroxide paste. Identification of selected bacterial taxa that are potential producers of the SCFAs under investigation was also performed.
Materials and Methods Subjects and Sample Taking Samples were taken from patients who had been referred for root canal treatment to the Department of Endodontics, Estacio de Sa University. Only single-rooted and single-canalled teeth from 18 adult patients (ages ranging from 20 to 39 years) with carious lesions, necrotic pulps confirmed by pulp tests, and clinical and radiographic evidence of asymptomatic apical periodontitis were included in this study. Exclusion criteria included teeth with gross carious lesions, teeth with root or crown fracture, teeth subjected to previous endodontic treatment, patients who received antibiotic therapy within the previous 3 months, symptomatic teeth, and patients with periodontal pockets deeper than 4 mm. The study was approved by the Ethics Committee at Estacio de Sa University, Rio de Janeiro, Brazil, and written informed consent was obtained from all the patients. Samples were taken from the root canals as follows. After the tooth crown was cleansed with pumice, a rubber dam was placed, and the tooth and the surrounding field were decontaminated by a protocol that used 3% hydrogen peroxide followed by 2.5% NaOCl solution (17). Complete access preparations were made by using sterile burs without water spray. The operative field, including the pulp chamber, was again swabbed with 2.5% NaOCl, which was then inactivated with sterile 5% sodium thiosulfate. Sterility control samples were taken from the tooth surface with sterile paper points. All the teeth included in the study had sterility control samples uniformly negative after polymerase chain reaction (PCR) with universal bacterial primers. The root canal was filled with 10 mmol/L Tris-HCl (pH 8.0), and a K-type file no. 15 was introduced up to approximately 1 mm short of the root apex, on the basis of radiographs, and used to gently file the canal walls. Afterwards, the fluid in the canal was aspirated by using a sterile disposable syringe and transferred to a cryotube. These sampling procedures were repeated several times until 100 mL sampled fluid was obtained. Sample was immediately frozen at 80 C. This initial root canal sample was called S1. Each root canal was instrumented at the same visit by using BioRaCe instruments (FKG Dentaire, La Chaux-de-Fonds, Switzerland), with the working length established 1 mm short of the radiographic apex. Master apical files ranged from BR5 (40/.04) to BR7 (60/.04), depending on both the root anatomy and the preoperative root canal diameter. Patency of the apical foramen was confirmed with #20 Ktype file throughout the procedures. Irrigation was performed with either 2.5% NaOCl (9 teeth) or 2% CHX (FGM, Joinvile, SC, Brazil) (9 teeth) by using disposable syringes and NaviTip needles (Ultradent, South Jordan, UT) inserted up to 4 mm short of the working length. Two milliliters of the irrigating solution (NaOCl or CHX) was used after each file size. After preparation, the root canal was dried and then flushed with 5 mL either 10% sodium thiosulfate solution or mixture of 0.07% lecithin, 0.5% Tween 80, and 5% sodium thiosulfate to neutralize 2
Provenzano et al.
any residual NaOCl or CHX, respectively. Postinstrumentation (S2) samples were taken from the root canals as described for S1 samples. Smear layer was removed by using 17% EDTA for 3 minutes. The canal was dried with paper points, medicated with Ca(OH)2 paste in camphorated paramonochlorophenol and glycerin, and placed in the canals by means of lentulo spiral fillers. A radiograph was taken to ensure proper placement of the paste in the canal, and the access cavity was filled with at least 4-mm thickness of temporary cement (Coltosol; Coltene/Whaledent Inc, Cuyahoga Falls, OH). The second appointment was scheduled 1 week later. At this time, the tooth was isolated with a rubber dam, the operative field was disinfected before and after removal of the temporary cement, NaOCl was neutralized, and a sterility control sample was taken. All these procedures were carried out as outlined previously. The Ca(OH)2 paste was rinsed out of the canal by using sterile saline solution and the master apical file. The root canal walls were gently filed, and a postmedication sample (S3) was taken from the canal as described for S1. The canal was filled with gutta-percha and Sealer 26 (Dentsply, Petropolis, RJ, Brazil) by using cold lateral compaction, the tooth was temporized with glass ionomer cement, and a permanent restoration was planned. All clinical procedures were conducted by one experienced endodontist (J.C.P.).
High-performance Liquid Chromatography Analysis Detection of SCFAs was performed directly in clinical samples. Fifty-microliter aliquots of each sample were diluted in 5 mmol/L H2SO4 at 1:2 ratio, followed by centrifugation at 10,000 rpm/10 min. Dilution of clinical samples was needed to reduce possible interferences related to the injection of complex samples in high-performance liquid chromatography (HPLC) and to keep samples at the detection limit, because they were highly concentrated and generated very intense peaks. Analysis were conducted in HPLC equipped with DGU-20A5 degasser, LC-20AT liquid chromatograph, SIL-20A auto sampler, CTO-20A column oven, RID-10A refractive index detector, SPD-M20 A diode array detector, FRC-10A fraction collector, and CBM-20A communications bus module (Shimadzu, Kyoto, Japan). Aminex HPX-87H column (300 7, 8 mm; Bio-Rad Laboratories Ltd, Hercules, CA) was used with a mobile phase composed of 5 mmol/L H2SO4 and a flow rate of 0.6 mL/min. Total run time was 30 minutes, and the RID was used for the analysis. The column oven was set to 50 C. Commercial standards of the analyzed fatty acids (Sigma-Aldrich, S~ao Paulo, SP, Brazil) were injected before the sample analysis. A total of 20 mL of each sample and standards was injected in the HPLC. Negative controls consisted of HPLC ultrapure water. Retention time and detection limits for each fatty acid targeted are depicted in Table 1. Experiments were run in duplicate. Real-time PCR Analysis DNA was extracted from a volume of 50 mL of each clinical sample by using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA), following the protocol recommended by the manufacturer. DNA extracts served as templates in a presence/absence real-time PCR assay for the detection of selected endodontic pathogens that are potential producers of the SCFAs targeted. The 16S rRNA gene-based PCR primers were specific for Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, members of the Dialister and Streptococcus genera, and the Actinobacteria phylum. Universal bacterial 16S rRNA gene-based primers were also used to serve as controls. PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on an ABI 7500 real-time JOE — Volume -, Number -, - 2015
Clinical Research samples, but it occurred in 1 S2 sample (CHX group) and 9 S3 samples (5 from the NaOCl group and 4 from the CHX group). Acetic acid was found in 1 post-treatment sample. Data are shown in Table 2.
TABLE 1. HPLC Data of the Analyzed Fatty Acids Fatty acid
Molecular formula
Retention time (min)
Detection limit (mmol/L)
Acetic acid Butyric acid Glycolic acid Isovaleric acid Lactic acid Maleic acid Propionic acid
C2H4O2 C4H8O2 C2H4O3 C5H10O2 C3H6O3 C4H4O4 C3H6O2
14.523 21.372 12.256 24.607 11.839 17.036 9.429
0.014 0.63 0.55 0.058 0.11 0.33 1.49
PCR instrument (Applied Biosystems) in a total reaction volume of 20 mL. Primers in a concentration of 1 mmol/L each and DNA extract volume of 2 mL were added to the PCR master mix in MicroAmp Optical 96well reaction plates. Plates were sealed, centrifuged, and subjected to amplification. Primer sequences and cycling conditions were as described and validated previously (18–23). Negative and positive controls were included in each batch of samples analyzed. Negative samples consisted of sterile ultrapure water instead of the sample. Positive controls consisted of DNA extracted from pure cultures of representative strains of the target species or groups, including F. nucleatum (ATCC 25586 and ATCC 10953), P. micra (ATCC 33270), P. endodontalis (ATCC 35406), P. alactolyticus (C11b-d), Streptococcus mutans (ATCC 25175), and Actinomyces species and Dialister invisus isolated in a previous study (24). Experiments were run in triplicate for all samples and controls, and specificity of the amplification was always determined with the use of melting curve analysis.
Data Analysis Prevalence of the fatty acids and target species was recorded as the percentage of cases examined. Attempts were made to correlate the SCFA found in the sample concomitantly with its potential producer species.
Real-time PCR Analysis Real-time PCR analysis by using universal 16S rRNA gene-based primers revealed that all S1 samples were positive for total bacteria. Overall, 12 of 17 S2 samples (71%) and 8 of 17 S3 samples (47%) still had detectable bacterial levels. In NaOCl group, 6 of 9 S2 samples (67%) and 4 of 9 S3 samples (44%) exhibited positive PCR results for bacteria, whereas respective figures for the CHX group were 6 of 8 S2 samples (75%) and 4 of 8 S3 samples (50%). All the target taxa were found in S1 samples (Table 3). The most prevalent were F. nucleatum (76%), members of the Actinobacteria phylum (71%), Streptococcus species (59%), and P. micra (53%). The target taxa were also found in at least 1 post-treatment sample. Gram-positive taxa were much more prevalent in S2 and S3 than the gram-negative taxa, with Streptococcus species as the most prevalent bacteria regardless of the irrigant used. Matching each SCFA with its potential producers in the same clinical sample demonstrated that of the 26 samples positive for butyric acid, F. nucleatum was found in 14, P. alactolyticus in 8, and P. endodontalis in 5. None of the target taxa that are known butyric acid producers were found in 8 samples positive for this fatty acid. Of the 12 samples positive for propionic acid, F. nucleatum and members of the Actinobacteria phylum were detected in 8 and P. endodontalis and Dialister species in 3 samples each. None of the potential propionic acid producers were found in 2 samples positive for this acid. Lactic acid was found in 10 samples, 3 of which had Actinobacteria, and 2 had P. micra and Streptococcus species. Five lactic acid–positive samples yielded none of the potential producers. Succinic acid was found in 2 samples, one presenting P. micra and the other Actinobacteria. In the sample positive for acetic acid, 3 potential producers were found: Actinobacteria, Streptococcus, and P. micra.
Results
Discussion
SFCA Analysis One sample from the CHX group was lost during preparation for HPLC analysis, and the case was excluded. HPLC screening revealed that butyric acid was the most common fatty acid in S1 samples (10 of 17 samples, 59%). It was also frequently found in S2 and S3 samples from both NaOCl and CHX groups. Propionic acid was detected in 7 S1 samples (41%) and also in post-treatment samples from both NaOCl and CHX groups. Of the other fatty acids targeted, only succinic acid was also found in S1. Lactic acid was not present in detectable levels in S1
SCFA are well-known metabolic end products of anaerobic bacteria. Several candidate endodontic pathogens produce SCFAs, and because they are usually arranged in biofilms, these substances are likely to accumulate in the canal. To the best of our knowledge, this is the first study to evaluate the occurrence of SCFAs directly in endodontic clinical samples before and after treatment. Butyric and propionic acids were frequently found in initial samples. This was somewhat expected because many of the candidate endodontic pathogens present in primary infections are anaerobic bacteria
TABLE 2. SFCAs in Infected Root Canals before (S1) and after Root Canal Preparation (S2) with Either NaOCl or CHX as the Irrigant and after Intracanal Medication with Calcium Hydroxide (S3) NaOCl (n = 9)
CHX (n = 8)
Total (n = 17)
Fatty acids
S1
S2
S3
S1
S2
S3
S1
S2
S3
Butyric acid Propionic acid Lactic acid Succinic acid Acetic acid Maleic acid Glycolic acid Isovaleric acid
5 (56)* 5 (56) ND ND ND ND ND ND
4 (44) 2 (22) ND 1 (11) 1 (11) ND ND ND
4 (44) 1 (11) 5 (56) ND ND ND ND ND
5 (63) 2 (25) ND 1 (13) ND ND ND ND
4 (50) ND 1 (13) ND ND ND ND ND
4 (50) 2 (25) 4 (50) ND ND ND ND ND
10 (59) 7 (41) ND 1 (6) ND ND ND ND
8 (47) 2 (12) 1 (6) 1 (6) 1 (6) ND ND ND
8 (47) 3 (18) 9 (53) ND ND ND ND ND
ND, not detected. *Number of positive cases (percentage).
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Short-chain Fatty Acids in Infected Canals
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Clinical Research TABLE 3. Selected Endodontic Pathogens in Infected Root Canals before (S1) and after Root Canal Preparation (S2) with Either NaOCl or CHX as the Irrigant and after Intracanal Medication with Calcium Hydroxide (S3) NaOCl (n = 9)
CHX (n = 8)
Total (n = 17)
Bacteria
S1
S2
S3
S1
S2
S3
S1
S2
S3
Total bacteria Streptococcus species Actinobacteria P. micra F. nucleatum P. alactolyticus P. endodontalis Dialister species
9 (100)* 4 (44) 4 (44) 8 (89) 6 (67) 1 (11) 5 (56) 4 (44)
6 (67) 6 (67) 3 (33) 5 (56) 2 (22) 1 (11) 1 (11) 0 (0)
4 (44) 3 (33) 2 (22) 4 (44) 1 (11) 1 (11) 1 (11) 0 (0)
8 (100) 6 (75) 8 (100) 1 (13) 7 (88) 7 (88) 3 (38) 1 (13)
6 (75) 5 (63) 5 (63) 0 (0) 3 (38) 3 (38) 0 (0) 1 (13)
4 (50) 3 (38) 4 (50) 0 (0) 1 (13) 3 (38) 0 (0) 0 (0)
17 (100) 10 (59) 12 (71) 9 (53) 13 (76) 8 (47) 8 (47) 5 (29)
12 (71) 11 (65) 8 (47) 5 (29) 5 (29) 4 (24) 1 (6) 1 (6)
8 (47) 6 (35) 6 (35) 4 (24) 2 (12) 4 (24) 1 (6) 0 (0)
*Number of positive cases (percentage).
known to produce these fatty acids in vitro. Both substances were also found in some samples taken after preparation by using either NaOCl or CHX as the irrigant. This indicates either that these SCFAs were not completely eliminated from the canals by preparation or that bacteria remaining in these canals were still metabolically active. Other SCFAs detected in this study included succinic and acetic acids, both in lower prevalences, and lactic acid. The main biological effects of SCFAs related to inflammation include toxicity to several cell types and effects on neutrophil chemotaxis, phagocytosis, and cytokine production. Butyric and propionic acids have been demonstrated to be cytotoxic to Vero cells (5, 25), which are a line of monkey cells used for many purposes including screening for toxicity of bacteria to mammalian cells. Butyric acid can inhibit T-cell proliferation (7) and induce apoptosis in monocytes, T and B lymphocytes, and epithelial cells (26–30). Butyric acid also induces production of reactive oxygen species and the impairment of cell growth in fibroblasts (31). Effects of SCFAs on chemotaxis and phagocytosis have also been reported. Butyric, succinic, and propionic acids stimulate neutrophil migration to inflamed sites and suppress the phagocytic capacity of phagocytes (7, 32, 33). Succinic acid at concentrations commonly found in clinical abscesses has been shown to impair the antimicrobial activity of neutrophils (34, 35). Butyric acid can stimulate the production of proinflammatory cytokines by monocytes and neutrophils (7, 30). All these biological activities and the detection of these SCFAs in samples from primary endodontic infections lend credence to the assertion that these molecules can play a causative role (along with other virulence factors) in apical periodontitis. All the bacterial taxa targeted in the real-time PCR assay have been frequently found in primary endodontic infections (36–39), which was confirmed in this study. Streptococci and Actinobacteria were more commonly detected in post-treatment samples. This is also in agreement with previous studies (20, 40–42). Selection of the target taxa was based on the fact that they are very prevalent in primary infections and are recognized in vitro producers of the SCFAs under investigation. For instance, butyric acid is a by-product of P. endodontalis, F. nucleatum, and P. alactolyticus, whereas propionic acid is produced by the former 2 species and members of the Dialister genus and Actinobacteria phylum. P. micra and Actinobacteria produce succinic acid. All the target species can generate acetic acid. In most cases, there was a correlation between the fatty acid detected and the presence of a target species that is a potential producer of that fatty acid. However, matching the acid to its producer did not occur in some cases. This is because endodontic infections are polymicrobial infections, and it would be virtually impossible to target all the potential producer species. Further studies that use broad-range microbial identification are required for a more profound correlative analysis.
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Particularly noteworthy was the high incidence of lactic acid in samples taken after intracanal medication with calcium hydroxide. Lactic acid is the most commonly recognized SCFA because of its occurrence at the termination of the anaerobic glycolytic cascade. In the oral environment, lactic acid is widely known because of its generation by oral bacteria in association with caries (43). Although this fatty acid was not detected in initial samples and was found in only 1 postinstrumentation sample, it was present in 9 post-medication samples. This may suggest a possible mechanism by which some bacteria respond to the alkaline stress induced by calcium hydroxide. Alternatively, the possibility also exists that lactic acid production is merely a characteristic of bacteria surviving the effects of calcium hydroxide. Potential lactic acid producers, such as Streptococcus species, Actinobacteria, and P. micra, were detected in several samples positive for this fatty acid. Other lactic acid producers not targeted in this study might well be present in these samples. This is especially applicable to the 5 lactic acid– positive samples in which none of the target species were found. Further studies should elaborate more on this increased detection of lactic acid after calcium hydroxide challenge and the producer species. In conclusion, this first report of the occurrence of SCFAs in infected root canals suggests that these bacterial products may play a role as virulence factors in the pathogenesis of apical periodontitis. These bacterial molecules were also found in post-treatment samples, always in association with bacterial presence. Significance of persistence of SCFAs after treatment and its effects on long-term outcome remain to be determined. Also, further quantitative studies are required to determine association of these substances with symptoms and ability of treatment to reduce their concentrations. The high occurrence of lactic acid after calcium hydroxide medication also deserves further investigations.
Acknowledgments This study was supported by grants from Fundac¸~ao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), Brazilian governmental institutions. The authors deny any conflicts of interest related to this study.
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