In 2009, Avila, Ojcius, and Yilmaz coauthored a paper entitled The Oral Microbiota: Living with a Permanent Guest. At this time oral microbiota is still metagenomically new and little work has been done with them. Avila et. al. discusses that the general clinical interests that were being sequenced included nasal, oral, urogenital, and skin microbiotas. The largest interest however, was in gut microbiota. The technologies being used to analyze ranged from shotgun sequencing to pyrosequencing, which was relatively new. From the techniques used with the microbiotas of interest, oral microbiotas began to be observed.
Since pyrosequencing was a new technique and the metagenomic focus was in other areas, the metagenomic analysis of oral microbiota began with a concentration on what the microbiota where do and their effects. As Avila et al. were conducting their study, many of the species found in the oral cavity were not culturable in lab due to technological impediments. “Because of these limitations, researchers have [began] by identifying and characterizing the microorganisms with the largest representation within the communities of healthy mouths: Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella, Treponema, Nisseria, Haemophilis, Eubacteria, Lactobacterium, Capnocytophaga, Eikenella, Lepto- trichia, Peptostreptococcus, Staphylococcus, and Propionibacterium (Jenkinson and Lamont, 2005; Wilson, 2005).” These microorganisms have a mutualistic relationship with the host while also protecting the host from pathogens. The microorganisms form a barrier, which blocks pathogenic organism from infecting and causing disease in the host.
Biofilms are an important aspect of the mouth. The biofilms are formed both from commensal organisms and pathogenic organisms. Avila et al. also noted that oral microbiota face a somewhat unique challenge. The host organisms have the option whether or not to be orally hygienic. Based on the host’s diet choice, teeth brushing, salivating, chewing habits, tounge movement, and other oral aggravation, the microorganisms can have different levels of habitation difficulties.
Sequencing all the genetic material of the oral microbiota was an issue to Avila et al. In addition to looking at the biofilms the group worked to characterize the microorganisms.
Pyrosequencing is a DNA sequencing method. It is also known as 454 Pyrosequencing due to it being licensed by 454 Life Sciences. Currently it costs between $5000-$7000 to use this method for a ~10 hour run sequence. DNA is sequenced by using emitted light during the incorporation of nucleotides. As a nucleotide is added to the forming sequences, the intensity of the light emitted changes. The nucleotides are tested for compatibility and if matched, they are added to the sequence. Otherwise the other nucleotides are tested. In the following, Wikipedia describes the process with more detail:
- The addition of one of the four deoxynucleoside triphosphates (dNTPs) (dATPαS, which is not a substrate for a luciferase, is added instead of dATP) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
- ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program.
- Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.
Using 454 pyrosequencing, researchers are now able to "compare the total genetic repertoire of the bacterial community" for subjects with varying health conditions and oral habits. In the study conducte by Belda-Ferre, Alcaraz, Cabrera-Rubio, Romero, Simon-Soro, Pignatelli, and Mira, 25 volenteers were used. The subjects were asked not to brush their teeth for 24hours and information was gathered about their "oral hygiene, diet, and signs of periodontal disease." DNA was also gathered from the subjects' mouths. During sequence analysis of the DNA, artificially replicated and human sequences were removed. The analysis found that in healthy individuals Bacilli and Gamma - Proteobacteria are more common, while in subjects with disease "anaerobic taxa like Clostridiales and Bacteroidetes are more frequent."