- Email: [email protected]
Prevalence of New Candidate Pathogens Prevotella baroniae, Prevotella multisaccharivorax and As-yet-uncultivated Bacteroidetes clone X083 in Primary Endodontic Infections
Clinical Research
Prevalence of New Candidate Pathogens Prevotella baroniae, Prevotella multisaccharivorax and As-yet-uncultivated Bacteroidetes clone X083 in Primary Endodontic Infections ˆ¸cas, PhD,* and Jose´ F. Siqueira Jr., PhD* Isabela N. Ro Abstract Introduction: Culture-independent studies have revealed a broad spectrum of oral bacterial taxa that may be associated with disease. This study investigated the prevalence of three new candidate oral pathogens: Prevotella baroniae, Prevotella multisaccharivorax, and as-yet-uncultivated Bacteroidetes oral clone X083 in primary endodontic infections using a devised culture-independent approach. Methods: Genomic DNA was isolated from samples taken from 52 teeth with different forms of apical periodontitis and used as template in a taxon-specific 16S rRNA gene-based nested polymerase chain reaction assay to determine the prevalence of the 3 target taxa. Results: Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax were respectively detected in 81%, 43%, and 38% of the root canals of teeth associated with chronic apical periodontitis, in 60%, 40% and 40% of the canals of teeth with acute apical periodontitis, and in 14%, 24%, and 5% of the pus aspirates from acute apical abscesses. No targeted taxon was positively associated with abscesses or clinical symptoms. Overall, Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax were found in 50%, 35%, and 25% of the samples taken from primary endodontic infections, respectively. Conclusions: Findings confirmed that the two newly named species and the uncultivated phylotype targeted in this study are associated with different forms of apical periodontitis, and a pathogenetic or at least an ecologic role is suspected. (J Endod 2009;35:1359–1362)
Key Words 16S Ribosomal RNA gene, acute apical abscess, apical periodontitis, endodontic infection, nested polymerase chain reaction
From the *Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Esta´cio de Sa´ University, Rio de Janeiro, RJ, Brazil. Address requests for reprints to Dr. Jose´ F. Siqueira Jr, Esta´cio de Sa´ University, Av Alfredo Baltazar da Silveira, 580/ cobertura, Recreio, Rio de Janeiro, Brazil 22790-710. E-mail address: [email protected]; [email protected] 0099-2399/$0 - see front matter Copyright ª 2009 American Association of Endodontists. doi:10.1016/j.joen.2009.05.033
JOE — Volume 35, Number 10, October 2009
A
n important step in the understanding of the pathogenesis of any infectious disease caused by mixed microbial communities is to provide detailed descriptions of the diversity of species involved, from high to low abundant species (1). Therefore, the microbiota associated with the diverse forms of apical periodontitis has been extensively investigated by culture-dependent and culture-independent approaches, and new candidate pathogens have surfaced as a consequence of these efforts (2). Bacterial species/phylotypes linked to endodontic infections fall into nine phyla. Of these, the phylum Bacteroidetes is one of the most widely represented (or diverse) in endodontic infections (3–7). Some of its members, notably from the genera Prevotella, Porphyromonas, and Tannerella, have been frequently found in association with different forms of apical periodontitis, including acute abscesses (8–14). The genus Prevotella includes moderately saccharolytic, bile-sensitive species formerly ascribed to the genus Bacteroides (15). There are currently about 20 oral Prevotella species with validly published names, but the genus diversity has been shown to be much higher in the oral cavity by several molecular studies (16, 17). Newly named species as Prevotella baroniae and Prevotella multisaccharivorax have been described and reported to occur in association with endodontic and periodontal diseases (12, 13, 18–20). P. baroniae is an obligately anaerobic, non–spore-forming, nonmotile, saccharolytic gram-negative coccoid or short bacilli. Cells of this species are usually 0.6 mm 0.6 to 2 mm, whereas some may be even 3 to 8 mm long (18). Colonies are 1.2 to 3.8 mm in diameter, circular, high convex, and opaque with a shiny gray periphery and an off-white center. Colony morphology varies from entire to undulate. The major end products include acetic and succinic acids, but minor amounts of isovaleric acid and isobutyric acid are also produced. Given the high similarity between the 16S rRNA gene sequence (>99%), P. baroniae may be synonymous with Prevotella clones PUS9.180 and E9_42-E4, which were first identified in symptomatic (21) and asymptomatic (4) endodontic infections, respectively. This species has been detected in the human oral cavity of patients with chronic apical periodontitis (22, 23), acute apical abscesses (3, 11, 12), and root canal–treated teeth with posttreatment disease (20, 24) in addition to periodontal infections and samples from periodontally healthy subjects (18). Prevotella multisaccharivorax is an obligately anaerobic, non–spore-forming, nonmotile, saccharolytic gram-negative rod (19). Cells are 0.8 2.5 to 8.3 mm, and colonies are 0.5 to 0.7 mm in diameter, gray to off-white gray, circular, entire, slightly convex, and smooth. The major end products are succinic and acetic acids, but minor amounts of isovaleric acid are also produced. It was originally isolated from subgingival biofilm from patients with chronic marginal periodontitis (19) but has been subsequently detected in samples from root caries (25), chronic apical periodontitis (3), acute apical abscesses (3), and root canal–treated teeth with posttreatment disease (20). Oral clone X083 is an as-yet-uncultivated phylotype from the Bacteroidetes phylum first described by Paster et al (17) in samples from marginal periodontitis and acute necrotizing ulcerative gingivitis. As a phylotype that remains to be cultivated, it is known only by a 16S rRNA gene sequence. It has more than 99% similarity to Bacteroidales oral clone MCE7_20, which was first identified in chronic endodontic
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Clinical Research infections (4), and is thereby very likely to be synonymous with this phylotype. Bacteroidetes clone X083 has been recently detected by molecular methods in association with chronic apical periodontitis (3, 13, 26, 27), acute apical abscesses (12, 28), and root canal–treated teeth with posttreatment disease (24). This study is in line with our efforts to investigate the prevalence of candidate oral and endodontic pathogens in endodontic infections by using a highly sensitive nested polymerase chain reaction (nPCR) approach (2, 29). Because Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax have been recently suggested to be potential endodontic pathogens (3, 12, 13), the present study aimed at determining the prevalence of these three members of the Bacteroidetes phylum in primary endodontic infections using a culture-independent molecular biology method arguably more sensitive than the methods used in previous studies that reported the association of these taxa with endodontic infections.
Material and Methods Samples were taken from patients who had been referred for root canal treatment or emergency treatment to the Department of Endodontics, Esta´cio de Sa´ University. Abscess samples were also taken from patients who were seeking emergency treatment in three hospitals in Rio de Janeiro. Only single-rooted teeth from adult patients (ages ranging from 18 to 74 years), all of them having carious lesions, necrotic pulps, and radiographic evidence of apical periodontitis, were included in this study. The study protocol was approved by the Ethics Committee of the Esta´cio de Sa´ University. Selected teeth showed an absence of periodontal pockets deeper than 4 mm. In general, 52 samples of endodontic infections were obtained. Most samples were also analyzed in a previous investigation (14). Teeth were grouped as follows: (1) 21 asymptomatic cases diagnosed as chronic apical periodontitis; (2) 10 cases diagnosed as acute apical periodontitis, which showed symptoms such as tenderness to percussion and/or palpation, provoked or spontaneous pain exacerbated by mastication, absence of pus in the canals, and no swelling; and (3) 21 cases diagnosed as acute apical abscesses, which showed pain and localized or diffuse swellings along with fever, lymphadenopathy, or malaise. No apparent communication from the abscess to the oral cavity or the skin surface was observed. In cases of chronic or acute apical periodontitis, samples were obtained from the root canals using sterile paper points. Abscesses were sampled by aspiration of purulent exudate from the swollen mucosa using a sterile syringe. Sampling procedures and DNA extraction protocol were as described previously (30). Reverse primers specific for Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax, were designed as follows: 16S rRNA gene sequences of each of the target bacteria were retrieved from the GenBank, aligned with the sequences of their nearest neighbors in the phylogenetic tree, and potential primers were designed from these areas. The BLAST-based algorithm (31) was used to verify their uniqueness. Primers were tested for specificity against purified DNA from a panel of oral species (32). Samples from previous studies (12, 13) that were positive for the target taxa using another molecular technique were amplified by PCR using the same primers and protocol described herein, and PCR products were sequenced to confirm specificity of the method. No cross-reactions were observed, and sequenced amplicons yielded the target taxa. For the analysis of the prevalence of the three taxa in primary endodontic infections, whole-genomic DNA extracts from clinical samples were used as templates in an nPCR protocol. In the first PCR reaction, a practically full-length 16S rRNA gene fragment was amplified 1360
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by using a pair of broad-range bacterial primers (Table 1). Aliquots of 5 mL of the DNA extracts were used as a template in 25 mL of a reaction mixture containing 0.5-mmol/L concentration of each universal primer, 2.5 mL of 10 PCR buffer (Fermentas, Burlington, Canada), 2 mmol/L MgCl2, 1.25 U of Taq DNA polymerase (Fermentas), and a 25-mmol/L concentration of each deoxyribonucleoside triphosphate (Biotools, Madrid, Spain). Afterwards, 2 mL of the universal reaction was used as a template in each of the nested taxon-specific reaction. The 16S rRNA gene-based primers are depicted in Table 1. They consisted of a broad-range forward primer and a taxon-specific reverse primer. The second PCR reaction used to assess the occurrence of the target taxa was performed in a 50 mL of reaction mixture containing 1mmol/L concentration of each primer, 5 mL of 10 PCR buffer, 2 mmol/L of MgCl2, 1.25 U of Taq DNA polymerase, and 0.2 mmol/L of each deoxyribonucleoside triphosphate. Positive controls consisted of samples already known to be positive in early tests. The specificity of these controls was previously confirmed through amplicon sequencing as described earlier. One negative control consisting of sterile ultrapure water instead of the sample was included for every five samples in all batches of samples analyzed. Preparations were amplified in a DNA thermocycler (Mastercycler Personal; Eppendorff, Hamburg, Germany). The PCR temperature profile for the universal reaction included an initial denaturation step at 97 C for 1 minute, followed by 26 cycles of a denaturation step at 97 C for 45 seconds, a primer annealing step at 55 C for 45 seconds, an extension step at 72 C for 90 seconds, and a final step of 72 C for 10 minutes. PCR cycling conditions for the second round of amplification included an initial denaturation step at 95 C for 2 minutes, and a touchdown PCR was performed as follows: the denaturing temperature of each cycle was performed at 95 C for 30 seconds. The annealing temperature was initially set at 60 C and then lowered 0.5 C every other cycle until it reached 55 C. Twenty-five additional cycles were performed at 55 C. Primer annealing was performed by using this scheme for 30 seconds, and primer extension was performed at 72 C for 1 minute. The final extension step was at 72 C for 10 minutes. PCR amplicons were separated by electrophoresis in a 1.5% agarose gel, stained with GelRed (Biotium, Hayward, CA), and viewed under ultraviolet transillumination. A 50-bp DNA ladder digest (Invitrogen, San Diego, CA) served as the molecular size standard. Representative amplicons from positive clinical samples were sequenced to check for specificity. The prevalence of the three target taxa was recorded as the percentage of the cases under investigation. The chi-square test with Yates correction and the two-tailed Fisher exact test were used to analyze the association between these bacterial taxa and abscesses or the overall occurrence of symptoms (joint cases of acute apical periodontitis and acute apical abscesses). Significance was established at 5% (p < 0.05).
Results All samples were positive for the presence of bacteria as shown by the generation of PCR amplicons of the expected size (1,500 bp) after the first round of PCR amplification using broad-range 16S rRNA gene primers. In addition to revealing the occurrence of bacteria in all examined cases, these positive results also showed the suitability of DNA for nPCR analysis and indicated the absence of significant inhibitors in the reaction mixture. Negative controls using sterile ultrapure water instead of sample yielded no PCR amplicons. To confirm the specificity of the protocol used for amplification of clinical samples, five representative PCR products for each target taxon, exhibiting the expected size, were sequenced and compared with the original sequences in the GenBank database. For Bacteroidetes clone JOE — Volume 35, Number 10, October 2009
Clinical Research TABLE 1. PCR Primers Used for Identification of 3 New Candidate Endodontic Pathogens Species in Primary Endodontic Infections Taxa
Primer sequences
Bacteroidetes clone X083
8-27
50 - ACT TGA GTG GAG GGT AGG - 30
149-132
0
Prevotella baroniae
0
5 - AGA GTT TGA TYM TGG CTC AG -3 *
8-27
50 - GGT TAT ACC CGA GTC GCG - 30
154-137
0
Prevotella multisaccharivorax
0
5 - AGA GTT TGA TYM TGG CTC AG -3 *
8-27
50 - CTT CAG ATG GCC TCA TAG G - 30
204-186
0
Broad-range 16S rRNA gene
Position (bp)
50 - AGA GTT TGA TYM TGG CTC AG -30 *
0
5 - AGA GTT TGA TYM TGG CTC AG - 3
8-27
50 - ACG GCT ACC TTG TTA CGA CTT - 30
1512-1492
*Broad-range forward primer.
X083, the level of similarity between the sequences obtained and the Bacteroidetes clone X083 sequence in GenBank was always 100%. For P. baroniae, the levels of similarity between the sequences ranged from 98.6% to 100%. For P. multisaccharivorax, similarity levels ranged from 98.5% to 99%. Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax were respectively detected in 81%, 43%, and 38% of the root canals associated with chronic apical periodontitis. In cases diagnosed as acute apical periodontitis, Bacteroidetes clone X083 was found in 60%, whereas both P. baroniae and P. multisaccharivorax were encountered in 40%. Of the three target taxa, P. baroniae was the most prevalent in abscess samples, being detected in 24% of the cases. Bacteroidetes clone X083 and P. multisaccharivorax were found in 14% and 5% of the acute apical abscesses, respectively. Because the 3 taxa were very prevalent in chronic cases, no significant association with clinical symptoms was disclosed. Actually, Bacteroidetes clone X083 was found significantly more in canals of teeth with chronic apical periodontitis than in abscess aspirates (p < 0.01) or symptomatic cases (p < 0.01). P. multisaccharivorax was also more associated with chronic apical periodontitis than with acute abscesses (p = 0.02). No other statistically significant differences were disclosed. Overall, taxon-specific nPCR allowed the detection of Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax in 50%, 35%, and 25% of the samples taken from primary endodontic infections, respectively (Table 2).
Discussion Overall, Bacteroidetes clone X083 was detected in 50% of the cases of primary infections, with the highest prevalence in cases of chronic apical periodontitis. This uncultivated phylotype is certainly one of the most prevalent as-yet-uncultivated taxon in endodontic infections (12, 13, 24). Clone X083 has been initially detected in endodontic infections in samples from teeth with chronic apical periodontitis but not from abscesses (3). A further study using the
reverse-capture checkerboard DNA-DNA hybridization technique analyzed a larger number of samples and revealed the occurrence of clone X083 in one half of the canals associated with chronic apical periodontitis (13), which is in agreement with the present findings. In that study (13), Bacteroidetes clone X083 was also the most prevalent taxon at high levels (above 105). However, another study using the same checkerboard methodology also detected this phylotype in cases of acute apical abscesses (12). Some strong positive associations have been observed for Bacteroidetes clone X083, most notably with Parvimonas micra, P. baroniae, Fusobacterium nucleatum, Selenomonas sputigena, and Peptostreptococcus anaerobius (12, 13). Clone X083 uncultivated phylotype has also been recently detected in samples from endodontic treatment failure examined by broad-range PCR and clone library analysis (24). The present findings join other studies to suggest that the Bacteroidetes clone X083 can be a candidate endodontic pathogen that has been previously underrated by inherent limitations of culturing approaches. Because this phylotype emerges as a potentially important endodontic pathogen, efforts should be directed toward its cultivation so that its phenotypic and pathogenic features can be appreciated. About one third of the samples surveyed in the present study were positive for the presence of P. baroniae. Although more prevalent in asymptomatic cases, P. baroniae was also detected in 24% of the abscessed cases. This newly named species has been recently linked to chronic apical periodontitis (4, 22, 23), acute apical abscesses (3, 11, 12, 21), and samples from treated teeth with persistent disease (20, 24). Data from these recent studies suggest that P. baroniae is actually one of the most prevalent Prevotella species in endodontic infections (3, 11–13). P. baroniae has been encountered in positive associations with Bacteroidetes clone X083, P. micra, Porphyromonas endodontalis, Dialister invisus, and Tannerella forsythia (12, 13). Further studies should focus on its pathogenicity and susceptibility to antimicrobial agents commonly used in root canal treatment or in the treatment of abscesses.
TABLE 2. Prevalence of Three New Candidate Endodontic Pathogens in Primary Infections as Revealed by 16S rRNA Gene-based nPCR Taxa
Chronic apical periodontitis
Acute apical periodontitis
Acute apical abscess
Total
Bacteroidetes clone X083 Prevotella baroniae Prevotella multisaccharivorax
17/21 (81)* 9/21 (43) 8/21 (38)
6/10 (60) 4/10 (40) 4/10 (40)
3/21 (14) 5/21 (24) 1/21 (5)
26/52 (50) 18/52 (35) 13/52 (25)
*Number of positive cases/number of samples examined (percentage).
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Clinical Research P. multisaccharivorax was detected in 25% of the samples of primary endodontic infections. This species was first detected in both symptomatic and asymptomatic endodontic infections by a molecular study using terminal restriction fragment length polymorphism analysis (3). Subsequent studies using larger sample sizes and another molecular technique confirmed the participation of this species in endodontic infections of teeth with chronic apical periodontitis and acute apical abscess but in low prevalence values (12, 13). The frequency in which P. multisaccharivorax was detected in cases of chronic apical periodontitis was much higher in this study (38%) as compared with a previous study using the reverse-capture checkerboard technique (9%) (13). A study also using the checkerboard technique detected this species in 18% of the samples from root canal–treated teeth of German patients (20). Despite being cultivable, both newly named P. baroniae and P. multisaccharivorax have been almost exclusively detected in endodontic infections by culture-independent molecular methods. The probable only exception was a culture study that isolated P. baroniae from abscess samples but identified it by 16S rRNA gene sequencing as Prevotella clone PUS9.180 (11). In conclusion, overall data from this study using a highly sensitive molecular approach indicated the occurrence of Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax in one half, about one third, and one fourth of the samples of primary endodontic infections, respectively. Although these taxa were mostly found in asymptomatic cases, involvement with symptomatic cases was also observed. Findings from the present study confirm that Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax are associated with different forms of apical periodontitis and give support to their inclusion in the list of candidate endodontic pathogens.
Acknowledgments The authors are grateful to Mr. Marlei Gomes da Silva for technical assistance.
References 1. Huse SM, Dethlefsen L, Huber JA, et al. Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genet 2008;4: e1000255. 2. Siqueira JF Jr., Roˆc¸as IN. Molecular analysis of endodontic infections. In: Fouad AF, ed. Endodontic microbiology. Ames, Iowa: Wiley-Blackwell, 2009:68–107. 3. Sakamoto M, Roˆc¸as IN, Siqueira JF Jr., et al. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol 2006;21: 112–22. 4. Munson MA, Pitt-Ford T, Chong B, et al. Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 2002;81: 761–6. 5. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257–62. 6. Gomes BP, Pinheiro ET, Gade-Neto CR, et al. Microbiological examination of infected dental root canals. Oral Microbiol Immunol 2004;19:71–6. 7. Le Goff A, Bunetel L, Mouton C, et al. Evaluation of root canal bacteria and their antimicrobial susceptibility in teeth with necrotic pulp. Oral Microbiol Immunol 1997;12:318–22. 8. Sassone LM, Fidel RA, Faveri M, et al. A microbiological profile of symptomatic teeth with primary endodontic infections. J Endod 2008;34:541–5.
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9. Siqueira JF Jr., Roˆc¸as IN. Bacteroides forsythus in primary endodontic infections as detected by nested PCR. J Endod 2003;29:390–3. 10. Gomes BP, Montagner F, Jacinto RC, et al. Polymerase chain reaction of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in primary endodontic infections. J Endod 2007;33:1049–52. 11. Khemaleelakul S, Baumgartner JC, Pruksakorn S. Identification of bacteria in acute endodontic infections and their antimicrobial susceptibility. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:746–55. 12. Siqueira JF Jr, Roˆc¸as IN. The microbiota of acute apical abscesses. J Dent Res 2009; 88:61–5. 13. Roˆc¸as IN, Siqueira JF Jr. Root canal microbiota of teeth with chronic apical periodontitis. J Clin Microbiol 2008;46:3599–606. 14. Siqueira JF Jr, Rocas IN, Silva MG. Prevalence and clonal analysis of Porphyromonas gingivalis in primary endodontic infections. J Endod 2008;34:1332–6. 15. Shah HN, Collins DM. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 1990;40:205–8. 16. Nadkarni MA, Caldon CE, Chhour K-L, et al. Carious dentine provides a habitat for a complex array of novel Prevotella-like bacteria. J Clin Microbiol 2004;42: 5238–44. 17. Paster BJ, Boches SK, Galvin JL, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770–83. 18. Downes J, Sutcliffe I, Tanner AC, et al. Prevotella marshii sp. nov. and Prevotella baroniae sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2005;55:1551–5. 19. Sakamoto M, Umeda M, Ishikawa I, et al. Prevotella multisaccharivorax sp. nov., isolated from human subgingival plaque. Int J Syst Evol Microbiol 2005;55: 1839–43. 20. Roˆc¸as IN, Hulsmann M, Siqueira JF Jr. Microorganisms in root canal-treated teeth from a German population. J Endod 2008;34:926–31. 21. Wade WG, Spratt DA, Dymock D, et al. Molecular detection of novel anaerobic species in dentoalveolar abscesses. Clin Infect Dis 1997;25(Suppl 2): S235–6. 22. Vickerman MM, Brossard KA, Funk DB, et al. Phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections. J Med Microbiol 2007;56:110–8. 23. Sakamoto M, Siqueira JF Jr, Roˆc¸as IN, et al. Bacterial reduction and persistence after endodontic treatment procedures. Oral Microbiol Immunol 2007;22: 19–23. 24. Sakamoto M, Siqueira JF Jr, Roˆc¸as IN, et al. Molecular analysis of the root canal microbiota associated with endodontic treatment failures. Oral Microbiol Immunol 2008;23:275–81. 25. Preza D, Olsen I, Aas JA, et al. Bacterial profiles of root caries in elderly patients. J Clin Microbiol 2008;46:2015–21. 26. Machado de Oliveira JC, Gama TG, Siqueira JF Jr., et al. On the use of denaturing gradient gel electrophoresis approach for bacterial identification in endodontic infections. Clin Oral Investig 2007;11:127–32. 27. Saito D, de Toledo Leonardo R, Rodrigues JLM, et al. Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries. J Med Microbiol 2006;55:101–7. 28. Jacinto RC, Gomes BP, Desai M, et al. Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography. Oral Microbiol Immunol 2007;22:403–10. 29. Siqueira JF Jr, Roˆc¸as IN. Exploiting molecular methods to explore endodontic infections: part 2—redefining the endodontic microbiota. J Endod 2005;31:488–98. 30. Siqueira JF Jr, Roˆc¸as IN, Rosado AS. Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by denaturing gradient gel electrophoresis fingerprinting approach. Oral Microbiol Immunol 2004;19:363–70. 31. Altschul SF, Madden TL, Scha¨ffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25: 3389–402. 32. Siqueira JF Jr, Roˆc¸as IN, Souto R, et al. Microbiological evaluation of acute periradicular abscesses by DNA-DNA hybridization. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:451–7.
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Prevalence of New Candidate Pathogens Prevotella baroniae, Prevotella multisaccharivorax and As-yet-uncultivated Bacteroidetes clone X083 in Primary Endodontic Infections ˆ¸cas, PhD,* and Jose´ F. Siqueira Jr., PhD* Isabela N. Ro Abstract Introduction: Culture-independent studies have revealed a broad spectrum of oral bacterial taxa that may be associated with disease. This study investigated the prevalence of three new candidate oral pathogens: Prevotella baroniae, Prevotella multisaccharivorax, and as-yet-uncultivated Bacteroidetes oral clone X083 in primary endodontic infections using a devised culture-independent approach. Methods: Genomic DNA was isolated from samples taken from 52 teeth with different forms of apical periodontitis and used as template in a taxon-specific 16S rRNA gene-based nested polymerase chain reaction assay to determine the prevalence of the 3 target taxa. Results: Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax were respectively detected in 81%, 43%, and 38% of the root canals of teeth associated with chronic apical periodontitis, in 60%, 40% and 40% of the canals of teeth with acute apical periodontitis, and in 14%, 24%, and 5% of the pus aspirates from acute apical abscesses. No targeted taxon was positively associated with abscesses or clinical symptoms. Overall, Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax were found in 50%, 35%, and 25% of the samples taken from primary endodontic infections, respectively. Conclusions: Findings confirmed that the two newly named species and the uncultivated phylotype targeted in this study are associated with different forms of apical periodontitis, and a pathogenetic or at least an ecologic role is suspected. (J Endod 2009;35:1359–1362)
Key Words 16S Ribosomal RNA gene, acute apical abscess, apical periodontitis, endodontic infection, nested polymerase chain reaction
From the *Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Esta´cio de Sa´ University, Rio de Janeiro, RJ, Brazil. Address requests for reprints to Dr. Jose´ F. Siqueira Jr, Esta´cio de Sa´ University, Av Alfredo Baltazar da Silveira, 580/ cobertura, Recreio, Rio de Janeiro, Brazil 22790-710. E-mail address: [email protected]; [email protected] 0099-2399/$0 - see front matter Copyright ª 2009 American Association of Endodontists. doi:10.1016/j.joen.2009.05.033
JOE — Volume 35, Number 10, October 2009
A
n important step in the understanding of the pathogenesis of any infectious disease caused by mixed microbial communities is to provide detailed descriptions of the diversity of species involved, from high to low abundant species (1). Therefore, the microbiota associated with the diverse forms of apical periodontitis has been extensively investigated by culture-dependent and culture-independent approaches, and new candidate pathogens have surfaced as a consequence of these efforts (2). Bacterial species/phylotypes linked to endodontic infections fall into nine phyla. Of these, the phylum Bacteroidetes is one of the most widely represented (or diverse) in endodontic infections (3–7). Some of its members, notably from the genera Prevotella, Porphyromonas, and Tannerella, have been frequently found in association with different forms of apical periodontitis, including acute abscesses (8–14). The genus Prevotella includes moderately saccharolytic, bile-sensitive species formerly ascribed to the genus Bacteroides (15). There are currently about 20 oral Prevotella species with validly published names, but the genus diversity has been shown to be much higher in the oral cavity by several molecular studies (16, 17). Newly named species as Prevotella baroniae and Prevotella multisaccharivorax have been described and reported to occur in association with endodontic and periodontal diseases (12, 13, 18–20). P. baroniae is an obligately anaerobic, non–spore-forming, nonmotile, saccharolytic gram-negative coccoid or short bacilli. Cells of this species are usually 0.6 mm 0.6 to 2 mm, whereas some may be even 3 to 8 mm long (18). Colonies are 1.2 to 3.8 mm in diameter, circular, high convex, and opaque with a shiny gray periphery and an off-white center. Colony morphology varies from entire to undulate. The major end products include acetic and succinic acids, but minor amounts of isovaleric acid and isobutyric acid are also produced. Given the high similarity between the 16S rRNA gene sequence (>99%), P. baroniae may be synonymous with Prevotella clones PUS9.180 and E9_42-E4, which were first identified in symptomatic (21) and asymptomatic (4) endodontic infections, respectively. This species has been detected in the human oral cavity of patients with chronic apical periodontitis (22, 23), acute apical abscesses (3, 11, 12), and root canal–treated teeth with posttreatment disease (20, 24) in addition to periodontal infections and samples from periodontally healthy subjects (18). Prevotella multisaccharivorax is an obligately anaerobic, non–spore-forming, nonmotile, saccharolytic gram-negative rod (19). Cells are 0.8 2.5 to 8.3 mm, and colonies are 0.5 to 0.7 mm in diameter, gray to off-white gray, circular, entire, slightly convex, and smooth. The major end products are succinic and acetic acids, but minor amounts of isovaleric acid are also produced. It was originally isolated from subgingival biofilm from patients with chronic marginal periodontitis (19) but has been subsequently detected in samples from root caries (25), chronic apical periodontitis (3), acute apical abscesses (3), and root canal–treated teeth with posttreatment disease (20). Oral clone X083 is an as-yet-uncultivated phylotype from the Bacteroidetes phylum first described by Paster et al (17) in samples from marginal periodontitis and acute necrotizing ulcerative gingivitis. As a phylotype that remains to be cultivated, it is known only by a 16S rRNA gene sequence. It has more than 99% similarity to Bacteroidales oral clone MCE7_20, which was first identified in chronic endodontic
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Clinical Research infections (4), and is thereby very likely to be synonymous with this phylotype. Bacteroidetes clone X083 has been recently detected by molecular methods in association with chronic apical periodontitis (3, 13, 26, 27), acute apical abscesses (12, 28), and root canal–treated teeth with posttreatment disease (24). This study is in line with our efforts to investigate the prevalence of candidate oral and endodontic pathogens in endodontic infections by using a highly sensitive nested polymerase chain reaction (nPCR) approach (2, 29). Because Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax have been recently suggested to be potential endodontic pathogens (3, 12, 13), the present study aimed at determining the prevalence of these three members of the Bacteroidetes phylum in primary endodontic infections using a culture-independent molecular biology method arguably more sensitive than the methods used in previous studies that reported the association of these taxa with endodontic infections.
Material and Methods Samples were taken from patients who had been referred for root canal treatment or emergency treatment to the Department of Endodontics, Esta´cio de Sa´ University. Abscess samples were also taken from patients who were seeking emergency treatment in three hospitals in Rio de Janeiro. Only single-rooted teeth from adult patients (ages ranging from 18 to 74 years), all of them having carious lesions, necrotic pulps, and radiographic evidence of apical periodontitis, were included in this study. The study protocol was approved by the Ethics Committee of the Esta´cio de Sa´ University. Selected teeth showed an absence of periodontal pockets deeper than 4 mm. In general, 52 samples of endodontic infections were obtained. Most samples were also analyzed in a previous investigation (14). Teeth were grouped as follows: (1) 21 asymptomatic cases diagnosed as chronic apical periodontitis; (2) 10 cases diagnosed as acute apical periodontitis, which showed symptoms such as tenderness to percussion and/or palpation, provoked or spontaneous pain exacerbated by mastication, absence of pus in the canals, and no swelling; and (3) 21 cases diagnosed as acute apical abscesses, which showed pain and localized or diffuse swellings along with fever, lymphadenopathy, or malaise. No apparent communication from the abscess to the oral cavity or the skin surface was observed. In cases of chronic or acute apical periodontitis, samples were obtained from the root canals using sterile paper points. Abscesses were sampled by aspiration of purulent exudate from the swollen mucosa using a sterile syringe. Sampling procedures and DNA extraction protocol were as described previously (30). Reverse primers specific for Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax, were designed as follows: 16S rRNA gene sequences of each of the target bacteria were retrieved from the GenBank, aligned with the sequences of their nearest neighbors in the phylogenetic tree, and potential primers were designed from these areas. The BLAST-based algorithm (31) was used to verify their uniqueness. Primers were tested for specificity against purified DNA from a panel of oral species (32). Samples from previous studies (12, 13) that were positive for the target taxa using another molecular technique were amplified by PCR using the same primers and protocol described herein, and PCR products were sequenced to confirm specificity of the method. No cross-reactions were observed, and sequenced amplicons yielded the target taxa. For the analysis of the prevalence of the three taxa in primary endodontic infections, whole-genomic DNA extracts from clinical samples were used as templates in an nPCR protocol. In the first PCR reaction, a practically full-length 16S rRNA gene fragment was amplified 1360
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by using a pair of broad-range bacterial primers (Table 1). Aliquots of 5 mL of the DNA extracts were used as a template in 25 mL of a reaction mixture containing 0.5-mmol/L concentration of each universal primer, 2.5 mL of 10 PCR buffer (Fermentas, Burlington, Canada), 2 mmol/L MgCl2, 1.25 U of Taq DNA polymerase (Fermentas), and a 25-mmol/L concentration of each deoxyribonucleoside triphosphate (Biotools, Madrid, Spain). Afterwards, 2 mL of the universal reaction was used as a template in each of the nested taxon-specific reaction. The 16S rRNA gene-based primers are depicted in Table 1. They consisted of a broad-range forward primer and a taxon-specific reverse primer. The second PCR reaction used to assess the occurrence of the target taxa was performed in a 50 mL of reaction mixture containing 1mmol/L concentration of each primer, 5 mL of 10 PCR buffer, 2 mmol/L of MgCl2, 1.25 U of Taq DNA polymerase, and 0.2 mmol/L of each deoxyribonucleoside triphosphate. Positive controls consisted of samples already known to be positive in early tests. The specificity of these controls was previously confirmed through amplicon sequencing as described earlier. One negative control consisting of sterile ultrapure water instead of the sample was included for every five samples in all batches of samples analyzed. Preparations were amplified in a DNA thermocycler (Mastercycler Personal; Eppendorff, Hamburg, Germany). The PCR temperature profile for the universal reaction included an initial denaturation step at 97 C for 1 minute, followed by 26 cycles of a denaturation step at 97 C for 45 seconds, a primer annealing step at 55 C for 45 seconds, an extension step at 72 C for 90 seconds, and a final step of 72 C for 10 minutes. PCR cycling conditions for the second round of amplification included an initial denaturation step at 95 C for 2 minutes, and a touchdown PCR was performed as follows: the denaturing temperature of each cycle was performed at 95 C for 30 seconds. The annealing temperature was initially set at 60 C and then lowered 0.5 C every other cycle until it reached 55 C. Twenty-five additional cycles were performed at 55 C. Primer annealing was performed by using this scheme for 30 seconds, and primer extension was performed at 72 C for 1 minute. The final extension step was at 72 C for 10 minutes. PCR amplicons were separated by electrophoresis in a 1.5% agarose gel, stained with GelRed (Biotium, Hayward, CA), and viewed under ultraviolet transillumination. A 50-bp DNA ladder digest (Invitrogen, San Diego, CA) served as the molecular size standard. Representative amplicons from positive clinical samples were sequenced to check for specificity. The prevalence of the three target taxa was recorded as the percentage of the cases under investigation. The chi-square test with Yates correction and the two-tailed Fisher exact test were used to analyze the association between these bacterial taxa and abscesses or the overall occurrence of symptoms (joint cases of acute apical periodontitis and acute apical abscesses). Significance was established at 5% (p < 0.05).
Results All samples were positive for the presence of bacteria as shown by the generation of PCR amplicons of the expected size (1,500 bp) after the first round of PCR amplification using broad-range 16S rRNA gene primers. In addition to revealing the occurrence of bacteria in all examined cases, these positive results also showed the suitability of DNA for nPCR analysis and indicated the absence of significant inhibitors in the reaction mixture. Negative controls using sterile ultrapure water instead of sample yielded no PCR amplicons. To confirm the specificity of the protocol used for amplification of clinical samples, five representative PCR products for each target taxon, exhibiting the expected size, were sequenced and compared with the original sequences in the GenBank database. For Bacteroidetes clone JOE — Volume 35, Number 10, October 2009
Clinical Research TABLE 1. PCR Primers Used for Identification of 3 New Candidate Endodontic Pathogens Species in Primary Endodontic Infections Taxa
Primer sequences
Bacteroidetes clone X083
8-27
50 - ACT TGA GTG GAG GGT AGG - 30
149-132
0
Prevotella baroniae
0
5 - AGA GTT TGA TYM TGG CTC AG -3 *
8-27
50 - GGT TAT ACC CGA GTC GCG - 30
154-137
0
Prevotella multisaccharivorax
0
5 - AGA GTT TGA TYM TGG CTC AG -3 *
8-27
50 - CTT CAG ATG GCC TCA TAG G - 30
204-186
0
Broad-range 16S rRNA gene
Position (bp)
50 - AGA GTT TGA TYM TGG CTC AG -30 *
0
5 - AGA GTT TGA TYM TGG CTC AG - 3
8-27
50 - ACG GCT ACC TTG TTA CGA CTT - 30
1512-1492
*Broad-range forward primer.
X083, the level of similarity between the sequences obtained and the Bacteroidetes clone X083 sequence in GenBank was always 100%. For P. baroniae, the levels of similarity between the sequences ranged from 98.6% to 100%. For P. multisaccharivorax, similarity levels ranged from 98.5% to 99%. Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax were respectively detected in 81%, 43%, and 38% of the root canals associated with chronic apical periodontitis. In cases diagnosed as acute apical periodontitis, Bacteroidetes clone X083 was found in 60%, whereas both P. baroniae and P. multisaccharivorax were encountered in 40%. Of the three target taxa, P. baroniae was the most prevalent in abscess samples, being detected in 24% of the cases. Bacteroidetes clone X083 and P. multisaccharivorax were found in 14% and 5% of the acute apical abscesses, respectively. Because the 3 taxa were very prevalent in chronic cases, no significant association with clinical symptoms was disclosed. Actually, Bacteroidetes clone X083 was found significantly more in canals of teeth with chronic apical periodontitis than in abscess aspirates (p < 0.01) or symptomatic cases (p < 0.01). P. multisaccharivorax was also more associated with chronic apical periodontitis than with acute abscesses (p = 0.02). No other statistically significant differences were disclosed. Overall, taxon-specific nPCR allowed the detection of Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax in 50%, 35%, and 25% of the samples taken from primary endodontic infections, respectively (Table 2).
Discussion Overall, Bacteroidetes clone X083 was detected in 50% of the cases of primary infections, with the highest prevalence in cases of chronic apical periodontitis. This uncultivated phylotype is certainly one of the most prevalent as-yet-uncultivated taxon in endodontic infections (12, 13, 24). Clone X083 has been initially detected in endodontic infections in samples from teeth with chronic apical periodontitis but not from abscesses (3). A further study using the
reverse-capture checkerboard DNA-DNA hybridization technique analyzed a larger number of samples and revealed the occurrence of clone X083 in one half of the canals associated with chronic apical periodontitis (13), which is in agreement with the present findings. In that study (13), Bacteroidetes clone X083 was also the most prevalent taxon at high levels (above 105). However, another study using the same checkerboard methodology also detected this phylotype in cases of acute apical abscesses (12). Some strong positive associations have been observed for Bacteroidetes clone X083, most notably with Parvimonas micra, P. baroniae, Fusobacterium nucleatum, Selenomonas sputigena, and Peptostreptococcus anaerobius (12, 13). Clone X083 uncultivated phylotype has also been recently detected in samples from endodontic treatment failure examined by broad-range PCR and clone library analysis (24). The present findings join other studies to suggest that the Bacteroidetes clone X083 can be a candidate endodontic pathogen that has been previously underrated by inherent limitations of culturing approaches. Because this phylotype emerges as a potentially important endodontic pathogen, efforts should be directed toward its cultivation so that its phenotypic and pathogenic features can be appreciated. About one third of the samples surveyed in the present study were positive for the presence of P. baroniae. Although more prevalent in asymptomatic cases, P. baroniae was also detected in 24% of the abscessed cases. This newly named species has been recently linked to chronic apical periodontitis (4, 22, 23), acute apical abscesses (3, 11, 12, 21), and samples from treated teeth with persistent disease (20, 24). Data from these recent studies suggest that P. baroniae is actually one of the most prevalent Prevotella species in endodontic infections (3, 11–13). P. baroniae has been encountered in positive associations with Bacteroidetes clone X083, P. micra, Porphyromonas endodontalis, Dialister invisus, and Tannerella forsythia (12, 13). Further studies should focus on its pathogenicity and susceptibility to antimicrobial agents commonly used in root canal treatment or in the treatment of abscesses.
TABLE 2. Prevalence of Three New Candidate Endodontic Pathogens in Primary Infections as Revealed by 16S rRNA Gene-based nPCR Taxa
Chronic apical periodontitis
Acute apical periodontitis
Acute apical abscess
Total
Bacteroidetes clone X083 Prevotella baroniae Prevotella multisaccharivorax
17/21 (81)* 9/21 (43) 8/21 (38)
6/10 (60) 4/10 (40) 4/10 (40)
3/21 (14) 5/21 (24) 1/21 (5)
26/52 (50) 18/52 (35) 13/52 (25)
*Number of positive cases/number of samples examined (percentage).
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Clinical Research P. multisaccharivorax was detected in 25% of the samples of primary endodontic infections. This species was first detected in both symptomatic and asymptomatic endodontic infections by a molecular study using terminal restriction fragment length polymorphism analysis (3). Subsequent studies using larger sample sizes and another molecular technique confirmed the participation of this species in endodontic infections of teeth with chronic apical periodontitis and acute apical abscess but in low prevalence values (12, 13). The frequency in which P. multisaccharivorax was detected in cases of chronic apical periodontitis was much higher in this study (38%) as compared with a previous study using the reverse-capture checkerboard technique (9%) (13). A study also using the checkerboard technique detected this species in 18% of the samples from root canal–treated teeth of German patients (20). Despite being cultivable, both newly named P. baroniae and P. multisaccharivorax have been almost exclusively detected in endodontic infections by culture-independent molecular methods. The probable only exception was a culture study that isolated P. baroniae from abscess samples but identified it by 16S rRNA gene sequencing as Prevotella clone PUS9.180 (11). In conclusion, overall data from this study using a highly sensitive molecular approach indicated the occurrence of Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax in one half, about one third, and one fourth of the samples of primary endodontic infections, respectively. Although these taxa were mostly found in asymptomatic cases, involvement with symptomatic cases was also observed. Findings from the present study confirm that Bacteroidetes clone X083, P. baroniae, and P. multisaccharivorax are associated with different forms of apical periodontitis and give support to their inclusion in the list of candidate endodontic pathogens.
Acknowledgments The authors are grateful to Mr. Marlei Gomes da Silva for technical assistance.
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