Year : 2020 | Volume
: 12 | Issue : 2 | Page : 110--114
Review: Endodontic Bacterial Characterization
Asha Kiran Panda1, Bibhuti Prasad Barik2,
1 Department of Zoology, GIET University; Department of Zoology, MM Mahavidyalaya, Berhampur, India
2 Department of Zoology, Khallikote Autonomous College, Berhampur, Odisha, India
Dr. Bibhuti Prasad Barik
Department of Zoology, Khallikote Autonomous College, Berhampur - 760 001, Odisha
Microorganisms, more specifically bacteria, are well known for endodontic pathogenesis. The motivation for quick identification of endodontic bacterial species is to inhibit their growth and to reduce the inflammation. Molecular characterization of these pathogens shall surely aid in understanding endodontics, preservation of oral health, and exploration of finest therapeutics. Characterizing endodontic bacteria structurally and functionally is crucial. Approaches such as microscopic, genotypic, and proteomic identification and molecular phylogeny have been effectively realistic to identify rapidly evolving endodontic strains. Developments of high-throughput DNA sequencing practices are believed to be having high success strategies in investigating community genome data following classical approaches. The existing dynamic metagenomic tools may be directed toward the development of innovative systems biology approaches for effective one-to-one therapeutic strategies.
|How to cite this article:|
Panda AK, Barik BP. Review: Endodontic Bacterial Characterization.J Int Clin Dent Res Organ 2020;12:110-114
|How to cite this URL:|
Panda AK, Barik BP. Review: Endodontic Bacterial Characterization. J Int Clin Dent Res Organ [serial online] 2020 [cited 2021 Apr 10 ];12:110-114
Available from: https://www.jicdro.org/text.asp?2020/12/2/110/303396
Correlation of endodontics and microbial pathogens is well known. Damage of teeth because of microbial intervention affects chewing, phonetics, oral patterns, tongue positioning, digestion, and nutrient assimilation. Although infections are reported as polymicrobial, bacteria are the most featured pathogens in the human tooth root canals. Kakehashi et al. (1966) have extensively highlighted bacterial reason of periapical illness. The existence of bacterial growth contributing endodontic problems is further studied by Möller et al. during 1981. Role of cytokines and neuropeptides has been established in the pulp tissues when bacterial lipopolysaccharides are present, leading to necrosis. Studies have also revealed that removal of bacteria before obturation has resulted better endodontic therapy. Bacteria are isolated frequently from the root canals of affected teeth during the failure of endodontic treatment. To understand the functional traits of endodontic microbiota, it is very essential to identify the specific species. The challenge of endodontic treatment is molecular characterization of the bacterial species, which may further facilitate the development of antimicrobial compounds for effective elimination of mutating strains.
Diversity of Endodontic Bacteria
Bacteria may reach the endodontic tissues by various ways such as dental tubules, exposed cavities, periodontal membrane, bloodstream via damaged tissues, and contamination by infectious tissues. Hundreds of intraradicular oral bacterial species have been reported, which are known to be Gram-negative anaerobic rods, Gram-positive cocci, and other bacterial species. The Gram-negative anaerobic rods mostly include Prevotella (Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, Prevotella multissacharivorax, Prevotella baroniae, and Prevotella denticola) and Porphyromonas (Porphyromonas endodontalis and Porphyromonas gingivalis) species; Tannerella forsythia; Dialister (Dialister pneumosintes and Dialister invisus) species; Fusobacterium (Fusobacterium nucleatum and Fusobacterium periodonticum); and spirochetes such as genus Treponema. Gram-positive anaerobic rods have also been recorded as endodontic pathogens such as Pseudoramibacter alactolyticus, Filifactor alocis, Actinomyces spp., Propionibacterium propionicum, Olsenella spp., Slackia exigua, Mogibacterium timidum, and Eubacterium spp. Gram-positive cocci namely Parvimonas micra, Streptococcus spp. (Streptococcus anginosus, Streptococcus mitis, and Streptococcus sanguinis), and Enterococcus faecalis have also been noted. Other bacterial spp. having endodontic presence are Campylobacter spp., Catonella morbic, Veillonella parvula, Eikenella corrodens, Granulicatella adiacens, Neisseria mucosa, Centipeda periodontii, Gemella morbillorum, Capnocytophaga gingivalis, Corynebacterium matruchotii, Bifidobacterium dentium, and anaerobic lactobacilli. Some other species are capable of crossing host defense barrier and are capable of extraradicular infections. These species are Actinomyces spp., P. propionicum, Treponema spp., P. endodontalis, P. gingivalis, Treponema forsythia, Prevotella spp., and F. nucleatum., E. faecalis are Gram-positive cocci, which may survive in the dental tubules for quite long period with persistent infections. Külekçi et al. studied aspirates of pus from dentoalveolar abscesses in patients who had received empirical antibiotic therapy within 1 month, and still, the predominant role of some anaerobic bacteria was noticed. Low abundant species may inhabit critical niches within intricate microbial community and may maintain the stability and virulence of a microbial community.
The growth culture of endodontic bacteria mostly reveals predominant living species and has played a strategic role in the association of precise endodontic infections. Bacterial growths have been observed on root canal specimens with electron microscopy, and the impact of ethylene diamine tetra acetic acid gel with Cetavlon has been assessed. Researchers have supported culture-dependent readings having benefits of establishing the bacterial burden, isolating bacterial strains for antibiotic susceptibility testing, and inspection of virulence dynamics. However, culture-based characterization protocols have frequently failed to replicate growth environment necessary by fastidious species with rigorous nutritional parameters. Hence, a number of pathogens are unmanageable. The traditional culture technique-based endodontic ecosystem database may not provide an exact idea of the microbial load because of failure of many pathogens to appear under routine laboratory conditions. Other shortcomings are difficulty in culturing large number of extant species, low-recovery rate of strains, delayed processing, low sensitivity/specificity, need of transport media, growth factors, toxicity of the culture, and metabolic dependency. This has led to culture-independent methods that are genotypic characterization. Because of the limitations of culture-based identification methods, relatively, new procedures have been established to identify microorganisms without culturing. Culture-independent, molecular analysis has revealed a more diverse microflora associated with endodontic infections than that revealed by cultural methods alone. Studies have revealed that persistent endodontic contaminations differ in their microflora. Major infections are usually anaerobic, Gram-negative bacteria, while Gram-positive facultative bacteria include persistent infections. Endodontic interactions and antibiotic resistance could be correlated, for example beta-lactamase positive-Prevotella species originate in dentoalveolar contaminations and the multidrug resistance is noticed E. faecalis.
Identification of bacteria has complicated culturing on solid culture media with painstaking, expensive, and time-consuming growth process. The disappointment of the culture process is sampling procedure and limitations in the finding the obligate anaerobic bacteria. In recent years, genetic methods have been increasingly approached which is giving promising results by allowing culturing difficult periodontal or endodontic pathogens. The study of nucleic acids has unlocked new opportunities for the identification of novel species and quick diagnostics. Molecular detection-based methods such as polymerase chain reaction (PCR) are intended to identify microbial DNA of the species. Universal probe and primers have been developed to estimate total bacteria by real-time PCR in the shortest possible time. This was advanced for the TaqMan system facilitating very sensitive identification of bacterial species defined in Bergey’s manual of determinative bacteriology, without cross-recognition of DNA from Eukarya or Archaea. A drawback of PCR was that it is unable differentiate between DNA from active or deceased cells, leading to confusion of living endodontic flora and past presence of pathogens in the root canal. False contaminations cannot be rule out in certain cases. Researchers have attempted to resolve bacterial diversity of the septic root canal by cloning, Sanger sequencing, and DNA fingerprinting, such as denaturing gradient gel and restriction fragment length polymorphism. These practices presented preliminary understandings of the bacterial diversity but could distinguish only predominant pathogen community species, while the benefit of next-generation sequencing (NGS) is the uncovering of little abundant strains. High-throughput technology allowed huge number of reads in single run with augmented sampling. Ling et al. described more than two hundred genera of ten phyla in oral/dental carries of children using high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis. NGS has been widely used to perceive bacterial diversity using 16S ribosomal RNA (16S rRNA) gene. Further, infection could be reduced if samples are screened and cloned with 16S rRNA sequencing. Brands et al. and others examined base analog inosine substitution at the 3’- terminus of broad-range 16S rRNA gene primers using terminal restriction fragment length polymorphism and clonal analysis and proposed combined use of inosine and unmodified primers intensifying the observed diversity of complex microbial communities. In humans, PCR detection of the human immunodeficiency virus (HIV) was confirmed by in situ hybridization of HIV genome in the dental pulp fibroblasts. Retrospective bacterial DNA diagnostic methods have been attempted in animals and human, having applications in forensic medicine and anthropology. Molecular approaches have attempted validation of complex endodontic microbiota than the simple single-culture approaches toward whole community pathogenicity. Tawfik et al. studied microbial diversity of endodontic infections using Illumina MiSeq sequencing platform in Egyptian patients, which was an essential footstep for the development of intracanal antimicrobial compounds. RNA sequencing studies centered on NGS are increasingly substituting microarrays, with greater sensitivity linking to dysbiotic routes of endodontic species. Transcriptomic analyses have offered a multitude of genes connected with biofilm creation by both Gram-positive and Gram-negative endodontic bacterial species. NGS attached reverse transcription-PCR offer huge parallel sequencing of nucleic acid fragments and period-efficient interpretation of whole genomes. Bioinformatic analytical pipelines have been developed for analyzing genomic and transcriptomic data. 16S rRNA marker gene survey methods have been recently developed where mixtures of environmental samples are useful for assessing analysis methods as one can evaluate methods based on calculated expected values using unmixed sample measurements and the mixture design.
Genomic and Phylogenetic Characterization
Whole-genomic DNA probes and checkerboard DNA–DNA hybridization analysis were initiated to evaluate the presence of root canal bacterial species. Metagenomic analysis of a healthy human dental plaque has been carried out, and ~ 2.8% of the total predicted genes coded for proteins having antibiotic resistance. A provisional taxonomic scheme was developed for the unnamed human oral bacterial isolates (which also included endodontic samples) and phylotypes and provided an online web-based platform, namely the human oral microbiome database.
Construction of genomic libraries has encouraged evolutionary case studies of endodontic strains. Computational phylogenetic tools having genomic foundation allow exploring evolutionary lineage and adaptation of endodontic bacteria. Genomic data are having advantage over phenotypic information as it is easy to relate sequence information more readily, reliably. Bacterial species sharing infected root canals might interact leading to horizontal exchange of antibiotic resistance, facilitating adoption of an optimal genetic profile for survival. Culture and molecular biology attempts have already shown that the endodontic bacterial community profiles differ in species richness in different types of infection. Genomic comparisons and phylogenetic analyses of E. faecalis were attempted by Zischka et al., citing the presence of novel genomic island. Yang et al. evaluated the phylogenetic sensitivity of the hypervariable regions of 16S rRNA genes.
The fast emerging proteomic tools have unlocked innovative ways to investigate the molecular basis of complex microbial pathogenesis. Microbial proteomics empowers protein annotation, comparative, semi-quantitative proteomics, protein localization, posttranslational modifications, and strain-committed proteogenomics. Proteomic characterization of bacterial virulence helps in understanding host-specific interactions. Liquid chromatography–tandem mass spectrometry (MS) is found to be good diagnostic platform to explore pathogenesis of root canal environment. The array of proteins expressed in endodontic infections reflects the intricate microbial existence having inflammatory dynamics. Surface-exposed virulence markers such as microbial surface components recognize adhesive matrix molecules and aggregation substances. Surface protein-mediated adherence to host tissues leads to the first steps of enterococcal colonization and subsequent biofilm formation, inflammation, and infection. Provenzano et al. evaluated the metaproteome of endodontic bacterial communities and reported numerous proteins linked to pathogenicity, resistance, adhesion, biofilm formation, antibiotic resistance, stress, exotoxins, invasins, proteases, abscesses, and metabolism-linked immune defense. Pérez-Llarena and Bou reviewed some rapid and reliable diagnostic techniques such as matrix-assisted laser desorption/ionization-time-of-flight MS that may be important for the diagnosis of bacterial resistance in clinical laboratories for characterizing resistant strains.
Enrichments of endodontic molecular tools along with bioinformatics possess the prospective to solve unanswered queries, but structural and functional characterization of the bacterial community still remains a challenge. Composite molecular characterization may substantially reduce diagnosis time and support in developing strategies to tackle proliferation trends.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
|1||Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germfree and conventional laboratory rats. J South Calif Dent Assoc 1966;34:449-51.|
|2||Möller AJ, Fabricius L, Dahlen G, Heyden G. Influence on periradicular tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981;89:475-84.|
|3||Hosoya S, Matsushima K. Stimulation of interleukin-1 beta production of human dental pulp cells by Porphyromonas endodontalis lipopolysaccharide. J Endod 1997;23:39-42.|
|4||Caviedes-Bucheli J, Muñoz HR, Azuero-Holguín MM, Ulate E. Neuropeptides in dental pulp: The silent protagonists. J Endod 2008;34:773-88.|
|5||Langeland K. Tissue response to dental caries. Endod Dent Traumatol 1987;3:149-71.|
|6||Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297-306.|
|7||Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent 2010;13:233-9.|
|8||Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 2006;42:80-7.|
|9||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.|
|10||Conrads G, Gharbia SE, Gulabivala K, Lampert F, Shah HN. The use of a 16s rDNA directed PCR for the detection of endodontopathogenic bacteria. J Endod 1997;23:433-8.|
|11||Dahle UR, Sunde PT, Tronstad L. Treponemas and endodontic infections. Endod Top 2003;6:160-70.|
|12||Sunde PT, Olsen I, Debelian GJ, Tronstad L. Microbiota of periapical lesions refractory to endodontic therapy. J Endod 2002;28:304-10.|
|13||Tronstad L, Kreshtool D, Barnett F. Microbiological monitoring and results of treatment of extraradicular endodontic infection. Endod Dent Traumatol 1990;6:129-36.|
|14||Külekçi G, Inanç D, Koçak H, Kasapoglu C, Gümrü OZ. Bacteriology of dentoalveolar abscesses in patients who have received empirical antibiotic therapy. Clin Infect Dis 1996;23 Suppl 1:S51-3.|
|15||Siqueira JF Jr., Rocas IN. Community as the unit of pathogenicity: An emerging concept as to the microbial pathogenesis of apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol 2009;107:870-8.|
|16||Somayaji K, Acharya SR, Bairy I, Prakash PY, Rao MS, Ballal NV. In vitro scanning electron microscopic study on the effect of doxycycline and vancomycin on enterococcal induced biofilm. Iran Endod J 2010;5:53-8.|
|17||Martins W Jr., De Rossi A, Rached RS, Rossi MA. A scanning electron microscopy study of diseased root surfaces conditioned with EDTA gel plus cetavlon after scaling and root planing. J Electron Microsc (Tokyo) 2011;60:167-75.|
|18||Fouad AF. Microbiological aspects of traumatic injuries. Dent Traumatol 2019;35:324-32.|
|19||Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG. Molecular and cultural analysis of the microfora associated with endodontic infections. J Dent Res 2002;81:761-6.|
|20||Jhajharia K. Microbiology of endodontic diseases: A review article. Int J Appl Dent Sci 2019;5:227-30.|
|21||Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 2002;148:257-66.|
|22||Ling Z, Kong J, Jia P, Wei C, Wang Y, Pan Z, et al. Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing. Microb Ecol 2010;60:677-90.|
|23||Siqueira JF Jr., Fouad AF, Rocas IN. Pyrosequencing as a tool for better understanding of human microbiomes. J Oral Microbiol 2012;4:10743.|
|24||Brands B, Vianna ME, Seyfarth I, Conrads G, Horz HP. Complementary retrieval of 16S rRNA gene sequences using broad range primers with inosine at the 3’ -terminus: Implications for the study of microbial diversity. FEMS Microbiol Ecol 2010;71:157-67.|
|25||Glick M, Trope M, Bagasra O, Pliskin ME. Human immunodeficiency virus infection of fibroblasts of dental pulp in seropositive patients. Oral Surg Oral Med Oral Pathol 1991;71:733-6.|
|26||Tawfik SA, Azab MM, Ahmed AA, Fayyad DM. Illumina MiSeq sequencing for preliminary analysis of microbiome causing primary endodontic infections in Egypt. Int J Microbiol 2018. doi: 10.1155/2018/2837328.|
|27||Magana M, Sereti C, Ioannidis A, Mitchell CA, Ball AR, Magiorkinis E, et al. Options and limitations in clinical investigation of bacterial biofilms. Clin Microbiol 2018; 31:e00084-16.|
|28||Olson ND, Kumar MS, Li S, Braccia DJ, Hao S, Timp W. A framework for assessing 16S rRNA marker-gene survey data analysis methods using mixtures. Microbiome 2020;8:8. [doi.org/10.1186/s40168-020-00812-1].|
|29||Xie G, Chain PS, Lo CC, Liu KL, Gans J, Merritt J, et al. Community and gene composition of a human dental plaque microbiota obtained by metagenomic sequencing. Mol Oral Microbiol 2010;25:391-405.|
|30||Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, et al. The human oral microbiome. J Bacteriol 2010;192:5002-17.|
|31||Zischka M, Künne CT, Blom J, Wobser D, Sakinç T, Schmidt-Hohagen KS, et al. Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40. BMC Genomics 2015. Available from: https://doi.org/10.1186/s12864-015-1367-x. [Last accessed on 2020 Jul 30].|
|32||Yang B, Wang Y, Qian PY. Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis. BMC Bioinfo 2016. Available from: https://doi.org/10.1186/s12859-016-0992-y. [Last accessed on 2020 Jul 30].|
|33||Schneider T, Riedel K. Environmental proteomics: Analysis of structure and function of microbial communities. Proteomics 2010;10:785-98.|
|34||Nandakumar R, Madayiputhiya N, Fouad AF. Proteomic analysis of endodontic infections by liquid chromatography-tandem mass spectrometry. Oral Microbiol Immunol 2009;24:347-52.|
|35||Provenzano JC, Siqueira JF Jr., Rôças IN, Domingues RR, Leme AD, Silva MR. Metaproteome analysis of endodontic infections in association with different clinical conditions. PLoS One 2013;8:E76108.|
|36||Pérez-Llarena FJ, Bou G. Proteomics as a tool for studying bacterial virulence and antimicrobial resistance. Front Microbiol 2016;7:410.|