Recent research in identification and quantification of periodontal pathogens

Introduction:

There has been a remarkable improvement in microbiological research in periodontics. The most basic approach is culture-dependent approach. Culture-dependent approaches are extremely useful for determining the antibiotic susceptibility of oral microbes and for assessing the pathogenicity of individual species. Periodontal plaque sample is allowed to grow in specific medias where the colonies of different species of micro-organisms can be identified. Disadvantage is that it requires experienced well trained labs and it is time consuming.

Presently we have culture independent approaches which involve RNA and DNA sequence identification techniques. Basically there are three main categories of molecular microbial analyses which include

  1. Polymerase chain reaction (PCR)-based methods (single target PCR, multiplex PCR and quantitative PCR),
  2. DNA–DNA hybridization methods (in situ hybridization, checkerboard hybridization and 16S ribosomal RNA-based microarrays) and
  3. Sequencing methods (latest, next generation sequencing techniques, such as pyrosequencing, real-time single-molecule DNA sequencing and nanopore-based sequencing).

Here is a brief description of currently used molecular microbial techniques

16S ribosomal RNA-based, reverse-capture oligonucleotide probes:

These are used to identify the microorganism species in plaque samples. 16S ribosomal RNA-based microarray is currently commercially available for diagnostic use. The ParoCheck® (Greiner bio-one, Frickenhausen, Germany) DNA chip targets 20 oral bacterial species and has been used to determine the microbial profiles of clinical samples. Recently high-density 16S ribosomal RNA-based microarrays have also been developed. The Phylochip, developed by the Affymetrix Corporation® (Santa Clara, CA) and Lawrence Berkeley Laboratories (Berkeley, CA) can detect up to 32,000 16S ribosomal RNA phylotypes1-3.

DNA Checkerboard hybridization:

This procedure involves extracting DNA from oral samples and hybridizing the sample against labeled probes representing whole genomes or 16S ribosomal RNA genes of known microbes. The method can be used to identify the subgingival plaque species in periodontal health and disease4-7.

Fingerprinting of amplified PCR products:

This method involves the digesting a mixture of PCR amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer8-10.

Quantitative PCR:

This method is used for quantifying the bacteria or genes in a given microbial plaque sample11-14.

Pyrosequencing:

It is based on the principal of sequence of enzyme triggered reactions that ultimately leads to the release of a pyrophosphate which in turn leads to generation of a luminescence signal after successful incorporation of a nucleotide into the DNA sequence15-16.

Microarrays:

They contain oligonucleotide probes that target RNA and DNA genes in biological samples17-20.

Research in any of above requires high end microbiological labs with highly trained personal which can give you accurate results without any error. A lot of gene based studies have been done. A thorough knowledge about the procedures involved is required before starting the study. 

References:

  1. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE. Bacterial diversity in human subgingival plaque. J Bacteriol 2001: 183: 3770–3783.
  2. Kroes I, Lepp PW, Relman DA. Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci USA 1999: 96: 14547–14552.
  3. Hutter G, Schlagenhauf U, Valenza G, Horn M, Burgemeister S, Claus H, Vogel U. Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens. Microbiology 2003: 149:67–75.
  4. Haffajee AD, Socransky SS. Relationship of cigarette smoking to the subgingival microbiota. J Clin Periodontol 2001: 28: 377–388.
  5. Darout IA, Albandar JM, Skaug N, Ali RW. Salivary microbiota levels in relation to periodontal status, experience of caries and miswak use in Sudanese adults. J Clin Periodontol 2002: 29: 411–420.
  6. Bostrom L, Bergstrom J, Dahle´n G, Linder LE. Smoking and subgingival microflora in periodontal disease. J Clin Periodontol 2001: 28: 212–219.
  7. Ximenez-Fyvie LA, Haffajee AD, Socransky SS. Comparison of the microbiota of supra- and subgingival plaque in subjects in health and periodontits. J Clin Periodontol 2000: 27: 648–657.
  8. Sakamoto M, Takeuchi Y, Umeda M, Ishikawa I, Benno Y. Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis. J Med Microbiol 2003: 52: 79–89.
  9. Liu WT, Marsh TL, Cheng H, Forney LJ. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 1997: 63: 4516–4522.
  10. Kitts CL. Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr Issues Intest Microbiol 2001: 2:17–25.
  11. Kawada M, Yoshida A, Suzuki N, Nakano Y, Saito T, Oho T, Koga T. Prevalence of Porphyromonas gingivalis in relation to periodontal status assessed by real-time PCR. Oral Microbiol Immunol 2004: 19: 289–292.
  12. Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, Arai H, Tanimoto I, Nishimura F, Takashiba S. Quantitative real-time PCR using Taq-Man and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol Med Microbiol 2003:39: 81–86.
  13. Morillo JM, Lau L, Sanz M, Herrera D, Silva A. Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. J Periodontal Res 2003: 38: 518–524.
  14. Nonnenmacher C, Dalpke A, Mutters R, Heeg K. Quantitative detection of periodontopathogens by real-time PCR. J Microbiol Methods 2004: 59: 117–125.
  15. Boyanton BL Jr, Luna RA, Fasciano LR, Menne KG, Versalovic J. DNA pyrosequencing-based identification of pathogenic Candida species by using the internal transcribed spacer 2 region. Arch Pathol Lab Med 2008: 132:667–674.
  16. Tuohy MJ, Hall GS, Sholtis M, Procop GW. Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp. Diagn Microbiol Infect Dis 2005: 51:245–250.
  17. Colombo AP, Boches SK, Cotton SL, Goodson JM, Kent R, Haffajee AD, Socransky SS, Hasturk H, Van Dyke TE, Dewhirst F, Paster BJ. Comparisons of subgingival microbial profiles of refractory periodontitis, severe periodontitis, and periodontal health using the human oral microbe identification microarray. J Periodontol 2009: 80: 1421–1432.
  18. Meka A, Bakthavatchalu V, Sathishkumar S, Lopez MC, Verma RK, Wallet SM, Bhattacharyya I, Boyce BF, Handfield M, Lamont RJ, Baker HV, Ebersole JL, Kesavalu L. Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles. Mol Oral Microbiol 2010: 25: 61–74.
  19. Preza D, Olsen I, Aas JA, Willumsen T, Grinde B, Paster BJ. Bacterial profiles of root caries in elderly patients. J Clin Microbiol 2008: 46: 2015–2021.
  20. Preza D, Olsen I, Willumsen T, Boches SK, Cotton SL, Grinde B, Paster BJ. Microarray analysis of the microflora of root caries in elderly. Eur J Clin Microbiol Infect Dis 2009: 28: 509–517.

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