Oral health, Microbes, DNA ConneXions, Dental health, Laboratory tests

Utilization of whole, fractioned, or ground teeth for DNA microbial testing. Is there a difference?

By Leslie Douglas, PhD, Blanche D. Grube, DMD, and Anita Vazquez Tibau

Introduction

The impact that oral health has on whole body health is no longer a question; in fact, over 100 systemic diseases have been linked to poor oral health.¹ According to the World Health Organization (WHO) almost half of the global population has an oral disease of some kind.² Oral diseases include dental decay, gum disease, and oral cancers. While oral diseases are largely preventable, tooth decay is the most common disease globally.³ What are now accepted as causative factors of oral cavities include oral microorganisms, oral environment, host, and time.⁴

The Center for Disease Control (CDC) has stated that periodontal disease affects almost 50% of those over 30 years old, and over 70% of those over 65 years old in the US. Statistically, periodontal disease is more common in men, those who are below the poverty level, those with less than a high school education, and current smokers.⁵ Observation of oral microbiota is a major indicator for the occurrence, development, and prognosis of disease. Oral bacteria can be used as a biomarker to potentially measure disease risk.⁶

In the early 1980s, Kary Mullis, invented the polymerase chain reaction (PCR) method. This groundbreaking discovery led him to receive the Noble Prize in Chemistry, in 1993.⁷ The use of PCR technology has become a mainstay in many disciplines and has been utilized in dentistry since its early stages. For example, in 1993 PCR was used to identify the periodontal pathogen Porphyromonas gingivalis in oral plaque samples.⁸ Porphyromonas gingivalis has been linked to various pathologies such as rheumatoid arthritis, cardiovascular pathologies, and neurodegenerative pathologies.⁹ Additionally, in 2005, open-ended PCRs were employed for genome mapping of the complete bacterial spectrum, leading to the development of the Human Oral Microbiome Database and CORE database.⁸ In forensics, PCR is able to make identifications utilizing dental DNA, owing to the stability of teeth in either extreme temperature changes or decomposition.¹⁰ PCR uses in dentistry include the following:

• Detection of periodontal pathogens,

• Detection of cariogenic pathogens,

• Detection of microorganisms involved with endodontic infections,

• Detection of viruses present in host cells: To detect human papillomavirus, hepatitis C virus, and other possible virus involvement in the etiology of periodontal disease,

• Detection of useful markers in diagnostic and prognostic of certain types of oral cancer,

• Quantitative estimates of various microorganisms.¹¹

According to Levy, the principle source of chronic degenerative disease starts in the oral cavity as follows:

• Root canal-treated teeth (always chronically infected),

• Other chronically infected teeth (often asymptomatic),

• Chronic periodontitis (gum inflammation and infection),

• Cavitational gangrene, at the sites of old extractions,

• Chronically infected tonsils, secondary to the chronic drainage of infected teeth,

• Infected dental implants, and

• Toxic metals, like mercury and nickel (not necessarily associated with infection).¹²

There is an abundance of bacteria found in all oral tissue, the most common being various Streptococcus species (oralis, mitis, and peroris).¹³

Oral streptococci are deemed an early colonizer representing over 80% of the initial makeup of oral biofilm, which can influence the health or disease of its host. Oral streptococci are divided into five different groups:

• Mutans group (prominent members are Streptococcus mutans and Streptococcus sobrinus),

• Salivarius group (Streptococcus salivarius),

• Anginosus group (Streptococcus anginosus and Streptococcus intermedius),

• Sanguinis group (Streptococcus sanguinis and Streptococcus gordonii),

• Mitis group (Streptococcus mitis and Streptococcus oralis).¹⁴

Many of these organisms from these five different groups have been discovered in the oral samples sent for PCR testing from patients who were suffering from the symptoms caused by these exact microorganisms.

A 2006 study by Nakano et al, discovered Streptococcus mutans was often identified in the heart valve (69%) and atheromatous plaque (74%) specimens, noting that Streptococcus mutans may be a causative factor of cardiovascular disease.¹⁵ Actinomyces are responsible for the development of dental plaque. These bacteria circulate into the intestinal tract and interact with other microbiota. These are a factor in assorted alimentary tract diseases such as inflammatory bowel disease and celiac disease.¹⁶ Lu et al. found that Prevotella intermedia presented in higher levels in gout and hyperuricemia patients. This Prevotella intermedia orignates in the oral cavity.¹⁷ The bacteria linked to periodontal health are Streptococcus, Granulicatella, Neisseria, Haemophilus, Corynebacterium, Rothia, Actinomyces, Prevotella, and Capnocytophaga.¹³ As mentioned above, Porphyromonas gingivalis was identified in the early use of PCR. Porphyromonas gingivalis is found in chronic periodontitis and has also been identified as a significant risk factor for developing Alzheimer’s disease.^9,18

Capnocytophaga species are deemed to be an opportunistic pathogen originating in the oral cavity. Capnocytophaga species have been found in many infections of immunocompromised hosts, including lung infections, cancer, bone infections, endocarditis, and sepsis in newborns.¹⁹

A paper by Yang et al. found that even though Corynebacterium sp is considered normal respiratory flora, it has been implicated in pneumonia, and may be more prevalent than previously thought. ²⁰

Fusobacterium nucleatum is a Gram-negative anaerobe found in the oral cavity; however, in disease states, it becomes dominant and can disseminate throughout the body. Fusobacterium nucleatum has been linked to the following:

• Oral infections (aggressive periodontitis, gingivitis, endodontic infections),

• Adverse pregnancy outcomes (chorioamnionitis, preterm birth, stillbirth, preeclampsia),

• GI disorders (colorectal cancer, inflammatory bowel disease, appendicitis),

• Other conditions (atherosclerosis, cerebral aneurysm, Lemierre’s syndrome, respiratory tract infections, organ abscesses, rheumatoid arthritis, Alzheimer’s).²¹

Root Canal Teeth

Root canal failure can be caused by many reasons such as:

• Persistence of bacteria (intra-canal and extra-canal),

• Inadequate filling of the canal (canals that are poorly cleaned and obturated),

• Overextensions of root filling materials,

• Improper coronal seal (leakage),

• Untreated canals (both major and accessory),

• Iatrogenic procedural errors such as poor access cavity design, and

• Complications of instrumentation (ledges, perforations, or separated instruments).

However, the most common cause of root canal failure is persistent bacterial infection.²²

Montalvo et el. investigated how Treponema denticola influenced disease progression in endodontically treated teeth. Treponema denticola is commonly found in root canals and may cause tissue damage. Due to its pervasiveness, it may be responsible for periapical bone lesions. The genus Treponema have been linked to numerous chronic diseases, such as syphilis (Treponema pallidum), periodontal diseases (including chronic periodontitis and acute necrotizing ulcerative gingivitis), endodontic infections, and some acute dental abscesses.²³

The pathogens of acute apical abscesses and asymptomatic root canal teeth were studied to evaluate the occurrence of the most common taxa using a two-step 16S rRNA gene-based PCR protocol. All samples found bacterial DNA. In abscesses, the most prevalent taxa were Fusobacterium nucleatum ssp. (60%), Porphyromonas endodontalis (53%), Parvimonas micra (51%), and Streptococcus species (45%).

The most frequently detected taxa in asymptomatic teeth were P. endodontalis (63%), Dialister invisus (58%), Olsenella uli (56%), and F. nucleatum ssp (51%).²⁴

A systematic review by Muacevic et al. also found that the principal reason for endodontic failure is due to bacteria, with the most common culprit being Enterococcus faecalis, noting that it more likely found in asymptomatic cases than symptomatic cases. Enterococcus faecalis was also found to be highly resistant to disinfectant treatments.²⁵ Microorganisms found in root canal treated teeth such as P. gingivalis, T. denticola, T. forsythia, and Solobacterium moorei have been connected with osteomyelitis, bacterial endocarditis, brain abscess, obesity, and preterm low birthweight. Root canal infections can develop into systemic infections.²⁶

Lačević et al explored the relationship between Tannerella forsythia, Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans at dual sites in concurrent endodontic- periodontal diseases. They found in the sixty samples from both the endodontium and periodontium the following:

• F. nucleatum ssp (100% of both endodontic and periodontal samples)

• T. forsythia (93% of periodontal pocket samples; 90% in primary endodontic infection)

• P. gingivalis (53% of periodontal pocket samples; 70% in primary endodontic infection)

• A. actinomycetemcomitans (13% of periodontal pocket samples; 3% in primary endodontic infection)

They concluded that the endodontic bacteria most likely originated from periodontal pockets and that endodontic infections maintained periodontal pockets and periodontal infection.²⁷

DNA ConneXions Oral Panel

The DNA ConneXions Oral Panel is a DNA PCR-based panel designed to detect the presence or absence of 88 microbes in oral samples. Sampling methods vary, inclusive of teeth, oral blood, bone, dental implants, Super Floss, etc. The question was raised if it is necessary to grind or otherwise fractionate whole tooth samples before employing our DNA extraction techniques. We hypothesized that a more robust and comprehensive microbial DNA extraction could be obtained by fractionating or grinding whole tooth samples. The objective of this study was to elucidate whether or not the porosity of teeth alone allows for the comprehensive release of the microorganisms of such solid samples.

Materials and Methods

The DNA extraction protocol employed at DNA ConneXions requires a 200 ul starting volume of sample. Blood, cavitations, and otherwise liquid samples submitted in volumes in excess of 200 ul are thoroughly homogenized and 200 ul is aliquoted and purified to obtain a prepared DNA sample. The volume of liquid samples received that are less than 200 ul are supplemented with Molecular Grade Water and are thoroughly homogenized; and 200 ul is aliquoted and utilized. Considering other sampling types (Super Floss, Paper Points, etc.), predetermined volumes of Molecular Grade Water are added, is thoroughly homogenized and 200 ul is obtained for testing/purification. Teeth, implants, either dry or accompanied by tissue, bone, blood, etc. are sampled in the same manner. The amount of Molecular Grade Water added to each sample is designed to supplement low volume/dry samples without compromising sample integrity.

Theoretically the addition of volume, followed by excessive agitation via vortex is thought to ‘dislodge’ the microbes present from the sample type provided, facilitating their transfer into the Molecular Grade Water, which is then drawn off and subjected to the DNA extraction protocol.

For DNA ConneXions, two juxtaposed samples were extracted from the same patient at the same time: #18 Non-vital tooth, #19 Root canal tooth. Each tooth was fractioned into two samples (Figure 1). One half of each sample was placed in sterile, single use plasticware that had been flushed with nitrogen gas to preserve genetic material during return shipping (provided by DNA ConneXions). The remaining half of each sample was ground into a fine powder using the KometaBio Dentin grinder (Fig. 2). Each grinding blade is a single use, sterile blade/collection cup, ensuring no cross contamination between samples occurs. The ground sample was also placed into a single use plasticware that has been flushed with Nitrogen gas. All samples were returned to DNA ConneXions for analysis, where two extractions for each sample was performed with the intent of comparing current and hypothesized methodologies.

Figure 1. Pre-Ground, Fractioned Samples #18, #19

Figure 2. Kometa BioGrinder

Each sample was subjected to the standard DNA ConneXions customary protocol for Oral Panel samples. Quantification via NanoDrop spectrophotometry was performed to ensure both nucleic acid purity and concentration. Samples were inoculated into prepared PCR plates encompassing the 88 microorganisms, included on the DNA ConneXions Oral Panel.

DNA extraction is performed utilizing a non-organic, spin column extraction protocol. DNA is precipitated with ethanol, and a series of ethanol-based washes are utilized, and purified DNA is collected and suspended in an elution buffer.

Purified samples are quantified for nucleic acid purity and concentration utilizing NanoDrop instrumentation. Acceptable concentration range is between 3-24 ng/ul, and acceptable 260/280 ratio is between 1.5-187. All four purified samples passed quantification.

Batches of 40 Oral Panel reaction plates are prepared at a time and stored at -20^oC. Each 96 well plate contains 88 unique sets of species-specific PCR primers, five positive controls and one negative control. A multichannel pipette is employed to dispense 3 ul of purified DNA per reaction well containing typical PCR reaction components. The plates are sealed with thermal films and amplified in Bioer Thermocyclers on a 23-step annealing gradient profile.

Amplification products are run on 1.2% agarose gels, with products ranging in size from 198bp – 1020bp, visualized with ultraviolet light and ethidium bromide stain.

Results

The DNA ConneXions Oral Panel report provides a general description, symptoms of infection, and CDC recommended treatment options for each organism detected. A Total Risk factor is calculated for each organism which considers the concentration of the organism present (Measured Risk Factor) and the characteristics of that organism (Pathogen Risk Factor). Pathogen Risk Factors are determined based on the overall characteristics of the organism: it’s overall pathogenicity and ability to cause disease, resistance to treatment, illness causing properties, links to systemic diseases, etc. The Measured Risk Factor is based on the visualized intensity of the amplification product, which has a direct linear correlation to the amount of the organismal starting material present in the sample.

Utilizing the proprietary DNA ConneXions Alpha-5 software, the combination of both the Measured Risk Factor and the Pathogen Risk Factor yields the Total Risk Factor of each organism detected. Figure 3 and Figure 4 graphically display both the whole and ground microbial flora detected with the DNA ConneXions Oral Panel, of #18 Nonvital Tooth and #19 Root Canal Tooth, respectively. Whole sample results are shown in red and ground sample results are shown in blue. The horizontal scale represents the Total Risk Factor for each microbe, which is based on the quantity of the organisms detected combined with the biological attributes of the organism.

CAPTION

Figure 3. DNA ConneXions Oral Panel Results for # 18 Nonvital Tooth (Fractioned and Ground Comparison)

Figure 4. DNA ConneXions Oral Panel Results for # 19 Root Canal Tooth (Fractioned and Ground Comparison)

While there are some differences seen between the amounts and identity of the organisms detected for each sampling type, the fractioned vs. ground results are well within the expected range we typically see for the DNA ConneXions Oral Panel, which is between 12-17 microbes detected. The slight differences could be due to the manipulation of the sample (ex. fractioning and/or grinding) and may have had an impact on the PCR results. Based on the number and concentration of microbes detected for the above sampling types, we determined that these variances are not statistically significant; and in general there was an overall consistency seen within the fractioned and ground sampling types, and the typically received whole tooth sampling type.

Site 18 Site 19 Site 18 and 19 Comparison

Fractioned (14) vs. ground (12) Fractioned (12) vs. ground (15) 1 unique microbe for site 18

10 microbes in common 10 microbes in common 2 unique microbes for site 19

4 unique for fractioned 2 unique for fractioned

2 unique for ground 5 unique for ground

Repeated and various samples from additional participants yielded consistent results with respect to total number of microbes detected, microbes in common between whole and crushed samples, and number of amplification products typically seen on the DNA Connexions Oral Panel (Table 1).

Table 1. Comparison of Number of Microbes Detected Between Fractioned and Ground Oral Samples

Conclusions

We have included research on some of the most common oral microbes that we commonly detect in utilizing the DNA ConneXions Oral Panel. The reports produced for both site 18 (nonvital tooth), 16 amplification products, and site 19 (root canal tooth), 17 amplification products, are typical Oral Panel reports. Our average number of amplification products spanning several thousand reports is between 12-17, with the highest ever at 52. As the crushed and fractioned samples yielded the same average number of results as previous whole tooth samples, it leads us to conclude that these sampling methods are equivalent and equally effective in the DNA extraction techniques utilized at DNA ConneXions.

We do not instruct dental practitioners to fraction or otherwise prepare their submitted tooth samples. Typically, we do not receive fractioned samples, unless it naturally occurs during surgical removal. It was possible that the fractionation and/or grinding of the tooth would yield more prolific results. However, based on the average number of amplification products achieved with whole, fractioned and ground samples, this hypothesis was negated. Had the fractioned and/or ground sample yielded far more amplification products it would have led us to remodel our theory on the aliquot protocol in place at DNA ConneXions. Rather, the ‘unremarkable’ and ‘average’ results suggest that the porosity of teeth allows for the transfer of the microbial flora present during the aliquot protocol, therefore, we were able to definitively show that it is not necessary to fraction or otherwise grind the sample in order to achieve an accurate representation of the microbial flora present. The importance of this type of DNA testing is evident, enabling us to assist both doctors and dentists by identifying potential pathogens that are linked to common diseases, using either sampling method. This insight can provide valuable information to aid in the implementation of appropriate interventions for the patient.

Special acknowledgement to Kelly J. Blodgett, DMD, NMD, IBDM of Blodgett Dental Care, Portland, Oregon. (503) 713-6980. info@bdcpdx.com.

References

1. Kane SF. The effects of oral health on systemic health. General Dentistry 2017; 411:35

2. The World Health Organization. Newsroom. March 25, 2020. https://www.who.int/news-room/fact-sheets/detail/oral-health

3. Peres MA, et al. Oral diseases: a global public health challenge. The Lancet. 2019; Vol 394

4. Chen X, et al. Microbial Etiology and Prevention of Dental Caries: Exploiting Natural Products to Inhibit Cariogenic Biofilms. Pathogens 2020; 9 (7): 569.

5. Centers for Disease Control and Prevention. Periodontal Disease. Available at: https://www.cdc.gov/oralhealth/conditions/periodontal-disease.html. Accessed October 17, 2020

6. Krishnan K, et al. A practical guide to the oral microbiome and its relation to health and disease. Oral Diseases. 2016; 23 (3)

7. Kary B. Mullis Facts. The Nobel Prize. 1993. https://www.nobelprize.org/prizes/chemistry/1993/mullis/facts/ . Accessed October 17, 2020

8. Maheaswari R, et al. Polymerase Chain Reaction: A Molecular Diagnostic Tool in Periodontology. J Indian Soc Periodontol. 2016 Mar-Apr; 20(2): 128–135.

9. Fiorillo L, et al, Porphyromonas gingivalis, Periodontal and Systemic Implications: A Systematic Review. Dent. J. 2019; 7(4): 114

10. Dineshkumar T. Role of Forensic Odontologist in Dentistry. Oral and Maxillofacial Pathology Journal. 2017;8(2):136-138

11. Patil SH, et al. Polymerase Chain Reaction: An Innovative Tool in Periodontal Diagnosis. World J Dent. 2013;4(1):60-66

12. Levy TE. Oral Pathogens: A Common Cause of Chronic Disease. JOM. 2016; 31(1) 7-18

13. Graves DT, et al. The Oral Microbiota Is Modified by Systemic Diseases. J Dent Res. 2019; 98 (2):148-156

14. Kreth J, et al. Bacterial and Host Interactions of Oral Streptococci. DNA Cell Biol. 2009; 28(8): 397-403

15. Nakano K, et al. Detection of Cariogenic Streptococcus mutans in Extirpated Heart Valve and Atheromatous Plaque Specimens. Journal of Clinical Microbiology. 2006; 3313-3317

16. Li J, et al. Actinomyces and Alimentary Tract Diseases: A Review of Its Biological Functions and Pathology. Biomed Res Int. 2018; 2018: 3820215.

17. Liu J, et al. Analysis of Oral Microbiota Revealed High Abundance of Prevotella Intermedia in Gout Patients. Cell Physiol Biochem. 2018; 49:1804–1812

18. Dominy SS, et al. Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Science Advances. 2019; 5(1): eaau3333.

19. Jolivet-Gougeon A. Capnocytophaga species. Antimicrobe. Available at: http://www.antimicrobe.org/b92.asp#r57. Accessed October 25, 2020

20. Yang K, et al. Corynebacteria as a cause of pulmonary infection: a case series and literature review. Pneumonia. 2018; 10 (10)

21. Han YW. Fusobacterium nucleatum ssp: a commensal-turned pathogen. Curr Opin Microbiol. 2015 Feb; 0: 141–147.

22. Tabassum S, et al. Failure of endodontic treatment: The usual suspects. Eur J Dent. 2016; 10(1): 144–147.

23. Alghamdi F, et al. Influence of Treponema denticola on apical periodontitis due to infection of endodontal origin. International of Applied Dental Sciences. 2019; 5(3): 172-175

24. Rocas IN, Siqueira Jr JF. Frequency and levels of candidate endodontic pathogens in acute apical abscesses as compared to asymptomatic apical periodontitis. Plos One. January 2, 2018.

25. Alghamdi F et al. The Influence of Enterococcus faecalis as a Dental Root Canal Pathogen on Endodontic Treatment: A Systematic Review. Cureus. 2020 Mar; 12(3): e7257.

26. Tiwari S, et al. Detection of Red complex bacteria, P. gingivalis, T. denticola and T. forsythia in infected root canals and their association with clinical signs and symptoms. J Family Medicine and Primary Care.2020; 9(4):1915-1950

27. Lačević et al. Correlation of Periodontal Pathogens in Concurrent Endodontic-Periodontal Diseases. Oral Biology and Dentistry 2015. Oral Biology and Dentistry. 2015.

Dr. Leslie J. Douglas completed her undergraduate studies in biology at the University of Hawaii at Hilo (UHH) before attending the University of Hawaii at Manoa (UHM), Department of Genetics and Molecular Biology. Currently, she is the principal investigator and laboratory manager of DNA ConneXions, a Colorado-based company focusing on bacterial, viral, fungal, and parasitic molecular-based detection assays. Her main focus is the research and development of a PCR-based Lyme test inclusive of Borrelia burgdorferi and a number of prevalent tick-borne disease co-infections, as well as the ongoing development of various molecular-based assays. Dr. Douglas’s research and patient demographics is yielding invaluable data to better understand the relationships between Lyme and other chronic conditions.

Blanche D. Grube graduated from Queens College, CUNY and received her doctorate from UMDNJ, now Rutgers School of Dental Medicine. She holds a second doctorate from Capital University of Integrative Medicine, Washington DC, and is a board-certified biological dentist and a past president of International Academy of Biological Dentistry and Medicine. She has lectured internationally on the Huggins-Grube Protocol. Besides holding several fellowships, she is the owner and CEO of DNA ConneXions, Biocomp Laboratories, Huggins Applied Healing, and Centers for Healing.

Anita Vazquez Tibau is a researcher at the Center for Environmental and Toxicological Research at the University of Puerto Rico. She has been working on a global ban on dental mercury for two decades, working with various non-governmental organizations. Since 2002, she has been an active participant in what is now called the Minamata Convention on Mercury treaty, where she wrote and delivered the closing statement on behalf of the International Academy of Oral Medicine and Toxicology. She has published articles in international journals, and several of her articles have been translated into multiple languages.  She has lectured in the US and abroad. Most recently she was the recipient of the Humanitarian Award at the Doctors Who Rock Gala, 2019, Orlando, Florida.