Root Microbiota in Primary and Secondary Apical Periodontitis

Serge Bouillaguet, Daniel Manoil, Myriam Girard, Justine Louis, Nadia Gaïa, Stefano Leo, Jacques Schrenzel, Vladimir Lazarevic, Serge Bouillaguet, Daniel Manoil, Myriam Girard, Justine Louis, Nadia Gaïa, Stefano Leo, Jacques Schrenzel, Vladimir Lazarevic

Abstract

Apical periodontitis is an inflammatory disease of the dental periradicular tissues triggered by bacteria colonizing necrotic root canals. Primary apical periodontitis results from the microbial colonization of necrotic pulp tissues. Secondary apical periodontitis results from a persistent infection of incorrectly treated root canals. The aim of this study was to characterize the microbiota present in primary and secondary intraradicular infections associated with apical periodontitis using 16S rRNA gene amplicon sequencing. Teeth exhibiting apical periodontitis with or without root canal treatment were extracted after informed consent. From each tooth, the intraradicular content as well as a dentin sample (control) were collected and subjected to DNA extraction. PCR amplicons of the V3-V4 region of the bacterial 16S rRNA gene were pooled and sequenced (2 × 300) on an Illumina MiSeq instrument. The bioinformatics analysis pipeline included quality filtering, merging of forward and reverse reads, clustering of reads into operational taxonomic units (OTUs), removal of putative contaminant OTUs and assigning taxonomy. The most prevalent and abundant OTU in both dentin and root canal samples was assigned to anaerobic bacterium Fusobacterium nucleatum. Multivariate analysis showed clustering of microbiota by sample type (dentin vs. intraradicular content) and, in root canals, by pathology (primary vs. secondary infection). The proportions of Enterococcus faecalis and F. nucleatum were, respectively, higher and lower when comparing secondary to primary infected root canals. Co-occurrence network analysis provided evidence of microbial interactions specific to the infection type. The identification of bacterial taxa differentially abundant in primary and secondary intraradicular infections may provide the basis for targeted therapeutic approaches aimed at reducing the incidence of apical periodontitis.

Keywords: 16S rRNA gene; Enterococcus faecalis; Fusobacterium nucleatum; apical periodontitis; community profiling; endodontics; oral microbiome.

Figures

FIGURE 1
FIGURE 1
Taxonomic composition and group average clustering of bacterial communities in root canal (A) and dentin (B). The heat map shows the relative abundance of OTUs across samples. OTUs with average relative abundance >0.5% in a given sample type are presented. The hierarchical clustering was based on square root-transformed proportions of OTUs and Bray–Curtis similarity matrix. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.
FIGURE 2
FIGURE 2
Principal coordinate analysis of Bray–Curtis similarity of bacterial communities. The analysis was based on square root-transformed proportions of OTUs and included either all (A), root canal (B), or dentin samples (C). PAP, primary apical periodontitis; SAP, secondary apical periodontitis.
FIGURE 3
FIGURE 3
OTUs differentially represented between PAP and SAP root samples. OTUs with significant changes (Wilcoxon rank-sum test, P < 0.05) and a minimum average relative abundance ≥0.5%) are presented. Boxplots show the first and third quartile (top and bottom edges of the rectangle) divided by median. Whiskers correspond to the highest and lowest values within 1.5× the interquartile range. Outliers are shown as circles. ∗0.01 < P < 0.05; ∗∗0.001 < P < 0.01; ∗∗∗P < 0.001. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.
FIGURE 4
FIGURE 4
Alpha diversity of the microbiota from root canal of PAP and SAP teeth. Shannon diversity index was based on OTU relative abundance. ∗∗0.001 < P < 0.01. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.
FIGURE 5
FIGURE 5
Co-occurrence and exclusion patterns among most abundant OTUs in PAP (A) and SAP (B) in root canal samples. OTUs found in at least eight samples and with a minimal average relative abundance of 1% (considering all root canal samples) were analyzed. The edges represent positive (blue) and negative (red) Spearman correlations. Only correlations with Spearman R > 0.5 or R < –0.5 are presented. The line thickness is proportional to the absolute value of the Spearman’s correlation. Node sizes reflect average relative abundance of each OTU. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.
FIGURE 6
FIGURE 6
Functional profiles of PAP and SAP-associated bacterial communities in root canal samples. The KEGG orthologs (predicted functions) inferred from the relative abundance of OTUs using PICRUSt were collapsed into level-3 KEGG ontology (KEGG pathways) and compared among PAP and SAP groups using STAMP. (A) Principal component analysis of KEGG pathways. (B) Significant differences (P < 0.05, two-sided Welch t-test) between PAP and SAP functions. STAMP was set to consider pathways represented by at least 10 sequences and an effect size filter (ratio of proportion) of 1.2. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.

References

    1. Anderson A. C., Hellwig E., Vespermann R., Wittmer A., Schmid M., Karygianni L., et al. (2012). Comprehensive analysis of secondary dental root canal infections: a combination of culture and culture-independent approaches reveals new insights. PLoS One 7:e49576. 10.1371/journal.pone.0049576
    1. Anderson A. C., Jonas D., Huber I., Karygianni L., Wölber J., Hellwig E., et al. (2015). Enterococcus faecalis from food, clinical specimens, and oral sites: prevalence of virulence factors in association with biofilm formation. Front. Microbiol. 6:1534. 10.3389/fmicb.2015.01534
    1. Anderson M. J., Willis T. J. (2003). Canonical analysis of principal coordinates: a useful method of constrained ordination for ecology. Ecology 84 511–525. 10.1890/0012-9658(2003)084[0511:CAOPCA];2
    1. Ando N., Hoshino E. (1990). Predominant obligate anaerobes invading the deep layers of root canal dentine. Int. Endod. J. 23 20–27. 10.1111/j.1365-2591.1990.tb00798.x
    1. Antunes H. S., Rôças I. N., Alves F. R. F., Siqueira J. F., Jr. (2015). Total and specific bacterial levels in the apical root canal system of teeth with post-treatment apical periodontitis. J. Endod. 41 1037–1042. 10.1016/j.joen.2015.03.008
    1. Bergenholtz G. (1974). Micro-organisms from necrotic pulp of traumatized teeth. Odontol. Revy 25 347–358.
    1. Bray R., Curtis J. T. (1957). An ordination of the upland forest communities of southern Wisconsin. Ecol. Monograph. 27 325–334. 10.2307/1942268
    1. Bullman S., Pedamallu C. S., Sicinska E., Clancy T. E., Zhang X., Cai D., et al. (2017). Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science 358 1443–1448. 10.1126/science.aal5240
    1. Caplan D. J., Chasen J. B., Krall E. A., Cai J., Kang S., Garcia R. I., et al. (2006). Lesions of endodontic origin and risk of coronary heart disease. J. Dent. Res. 85 996–1000. 10.1177/154405910608501104
    1. Chávez de Paz L. (2005). Gram-positive organisms in endodontic infections. Endod. Top. 9 79–96. 10.1111/j.1601-1546.2004.00107.x
    1. Chávez de Paz L. E., Bergenholtz G., Dahlén G., Svensäter G. (2007). Response to alkaline stress by root canal bacteria in biofilms. Int. Endod. J. 40 344–355. 10.1111/j.1365-2591.2006.01226.x
    1. Chen T., Yu W.-H., Izard J., Baranova O. V., Lakshmanan A., Dewhirst F. E. (2010). The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database 2010:baq013. 10.1093/database/baq013
    1. Deng Z.-L., Szafrański S. P., Jarek M., Bhuju S., Wagner-Döbler I. (2017). Dysbiosis in chronic periodontitis: key microbial players and interactions with the human host. Sci. Rep. 7:3703. 10.1038/s41598-017-03804-8
    1. Distel J. W., Hatton J. F., Gillespie M. J. (2002). Biofilm formation in medicated root canals. J. Endod. 28 689–693. 10.1097/00004770-200210000-00003
    1. Edgar R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26 2460–2461. 10.1093/bioinformatics/btq461
    1. Edgar R. C. (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10 996–998. 10.1038/nmeth.2604
    1. Evans M., Davies J. K., Sundqvist G., Figdor D. (2002). Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int. Endod. J. 35 221–228. 10.1046/j.1365-2591.2002.00504.x
    1. Fabricious L., Dahlen G., ÖHman A. E., MÖLler A. J. R. (1982). Predominant indigenous oral bacteria isolated from infected root canals after varied times of closure. Eur. J. Oral Sci. 90 134–144. 10.1111/j.1600-0722.1982.tb01536.x
    1. Faust K., Sathirapongsasuti J. F., Izard J., Segata N., Gevers D., Raes J., et al. (2012). Microbial Co-occurrence relationships in the human microbiome. PLoS Comput. Biol. 8:e1002606. 10.1371/journal.pcbi.1002606
    1. Hong B.-Y., Lee T.-K., Lim S.-M., Chang S. W., Park J., Han S. H., et al. (2013). Microbial analysis in primary and persistent endodontic infections by using pyrosequencing. J. Endod. 39 1136–1140. 10.1016/j.joen.2013.05.001
    1. Kakehashi S., Stanley H. R., Fitzgerald R. J. (1965). The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg. Oral Med. Oral Pathol. 20 340–349. 10.1016/0030-4220(65)90166-0
    1. Keijser B. J., Zaura E., Huse S. M., van der Vossen J. M., Schuren F. H., Montijn R. C., et al. (2008). Pyrosequencing analysis of the oral microflora of healthy adults. J. Dent. Res. 87 1016–1020. 10.1177/154405910808701104
    1. Keskin C., Demiryürek E. Ö., Onuk E. E. (2017). Pyrosequencing analysis of cryogenically ground samples from primary and secondary/persistent endodontic infections. J. Endod. 43 1309–1316. 10.1016/j.joen.2017.03.019
    1. Khalighinejad N., Aminoshariae M. R., Aminoshariae A., Kulild J. C., Mickel A., Fouad A. F. (2016). Association between systemic diseases and apical periodontitis. J. Endod. 42 1427–1434. 10.1016/j.joen.2016.07.007
    1. Langille M. G. I., Zaneveld J., Caporaso J. G., McDonald D., Knights D., Reyes J. A., et al. (2013). Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat. Biotechnol. 31 814–821. 10.1038/nbt.2676
    1. Lazarevic V., Gaïa N., Girard M., Schrenzel J. (2016). Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR. BMC Microbiol. 16:73. 10.1186/s12866-016-0689-4
    1. Lazarevic V., Whiteson K., Huse S., Hernandez D., Farinelli L., Østerås M., et al. (2009). Metagenomic study of the oral microbiota by Illumina high-throughput sequencing. J. Microbiol. Methods 79 266–271. 10.1016/j.mimet.2009.09.012
    1. Lleò M. M., Bonato B., Tafi M. C., Signoretto C., Boaretti M., Canepari P. (2002). Resuscitation rate in different enterococcal species in the viable but non-culturable state. J. Appl. Microbiol. 91 1095–1102. 10.1046/j.1365-2672.2001.01476.x
    1. Mayanagi G., Sato T., Shimauchi H., Takahashi N. (2004). Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects. Oral Microbiol. Immunol. 19 379–385. 10.1111/j.1399-302x.2004.00172.x
    1. McDonald D., Price M. N., Goodrich J., Nawrocki E. P., DeSantis T. Z., Probst A. (2012). An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6 610–618. 10.1038/ismej.2011.139
    1. Munson M. A., Pitt-Ford T., Chong B., Weightman A., Wade W. G. (2002). Molecular and Cultural analysis of the microflora associated with endodontic infections. J. Dent. Res. 81 761–766. 10.1177/0810761
    1. Ogata H., Goto S., Sato K., Fujibuchi W., Bono H., Kanehisa M. (1999). KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 27 29–34. 10.1093/nar/27.1.29
    1. Orstavik D., Kerekes K., Eriksen H. M. (1986). The periapical index: a scoring system for radiographic assessment of apical periodontitis. Endod. Dent. Traumatol. 2 20–34. 10.1111/j.1600-9657.1986.tb00119.x
    1. Özok A. R., Persoon I. F., Huse S. M., Keijser B. J. F., Wesselink P. R., Crielaard W., et al. (2012). Ecology of the microbiome of the infected root canal system: a comparison between apical and coronal root segments. Int. Endod. J. 45 530–541. 10.1111/j.1365-2591.2011.02006.x
    1. Parks D. H., Tyson G. W., Hugenholtz P., Beiko R. G. (2014). STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics 30 3123–3124. 10.1093/bioinformatics/btu494
    1. Paster B. J., Boches S. K., Galvin J. L., Ericson R. E., Lau C. N., Levanos V. A., et al. (2001). Bacterial diversity in human subgingival plaque. J. Bacteriol. 183 3770–3783. 10.1128/jb.183.12.3770-3783.2001
    1. Rôças I. N., Alves F. R. F., Rachid C. T. C. C., Lima K. C., Assunção I. V., Gomes P. N., et al. (2016). Microbiome of deep dentinal caries lesions in teeth with symptomatic irreversible pulpitis. PLoS One 11:e0154653. 10.1371/journal.pone.0154653
    1. Rôças I. N., Neves M. A. S., Provenzano J. C., Siqueira J. F. (2014). Susceptibility of as-yet-uncultivated and difficult-to-culture bacteria to chemomechanical procedures. J. Endod. 40 33–37. 10.1016/j.joen.2013.07.022
    1. Rôças I. N., Siqueira J. F. (2012). Characterization of microbiota of root canal-treated teeth with posttreatment disease. J. Clin. Microbiol. 50 1721–1724. 10.1128/jcm.00531-12
    1. Schloss P. D., Westcott S. L., Ryabin T., Hall J. R., Hartmann M., Hollister E. B., et al. (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75 7537–7541. 10.1128/AEM.01541-09
    1. Shannon P., Markiel A., Ozier O., Baliga N. S., Wang J. T., Ramage D., et al. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13 2498–2504. 10.1101/gr.1239303
    1. Siqueira J. F., Alves F. R. F., Rôças I. N. (2011). Pyrosequencing analysis of the apical root canal microbiota. J. Endod. 37 1499–1503. 10.1016/j.joen.2011.08.012
    1. Siqueira J. F., de Uzeda M. (1996). Disinfection by calcium hydroxide pastes of dentinal tubules infected with two obligate and one facultative anaerobic bacteria. J. Endod. 22 674–676. 10.1016/S0099-2399(96)80062-8
    1. Siqueira J. F., Rôças I. N. (2017). “The oral microbiota in health and disease: an overview of molecular findings,” in Oral Biology: Molecular Techniques and Applications eds Seymour G. J., Cullinan M. P., Heng N. C. K. (New York, NY: Springer; ) 127–138.
    1. Stuart C. H., Schwartz S. A., Beeson T. J., Owatz C. B. (2006). Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J. Endod. 32 93–98. 10.1016/j.joen.2005.10.049
    1. Sundqvist G. (1992). Associations between microbial species in dental root canal infections. Oral Microbiol. Immunol. 7 257–262. 10.1111/j.1399-302X.1992.tb00584.x
    1. Sundqvist G., Figdor D., Persson S., Sjögren U. (1998). Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 85 86–93. 10.1016/S1079-2104(98)90404-8
    1. Tyson G. H., Sabo J. L., Rice-Trujillo C., Hernandez J., McDermott P. F. (2018). Whole-genome sequencing based characterization of antimicrobial resistance in Enterococcus. Pathog. Dis. 76:fty018. 10.1093/femspd/fty018
    1. Tzanetakis G. N., Azcarate-Peril M. A., Zachaki S., Panopoulos P., Kontakiotis E. G., Madianos P. N., et al. (2015). Comparison of bacterial community composition of primary and persistent endodontic infections using pyrosequencing. J. Endod. 41 1226–1233. 10.1016/j.joen.2015.03.010
    1. Videvall E., Strandh M., Engelbrecht A., Cloete S., Cornwallis C. K. (2017). Direct PCR offers a fast and reliable alternative to conventional DNA isolation methods for gut microbiomes. mSystems 2:e00132-17. 10.1128/mSystems.00132-17
    1. Yoon S.-H., Ha S.-M., Kwon S., Lim J., Kim Y., Seo H., et al. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67 1613–1617. 10.1099/ijsem.0.001755
    1. Zehnder M., Guggenheim B. (2009). The mysterious appearance of enterococci in filled root canals. Int. Endod. J. 42 277–287. 10.1111/j.1365-2591.2008.01537.x
    1. Zhang J., Kobert K., Flouri T., Stamatakis A. (2014). PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30 614–620. 10.1093/bioinformatics/btt593

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