Role of Protective Species in Inhibiting

Virulence Factor in S. mutans

Monocultures Cocultures
Figure 4: Representative monocultures of PS and S. mutans Colorimetric Analysis
Affecting over 2.4 billion people worldwide, dental caries or tooth decay PS S. mutans and cocultures of the two species under different conditions
is the most prevalent disease globally. Caries is primarily caused by
Incubate monocultures of protective species aerobically and Acid Intensity in S. mutans 140 cultures Acid Intensity in S. mutans 159 Cultures
(aerobic, 5-8% CO2, microaerophilic, anaerobic).
Streptococcus mutans, a bacteria species that grows on enamel surfaces S. mutans anaerobically for 48 hours at 37 degrees Celsius 180 180
and produces lactic acid as part of its metabolic processes. As a result, Aerobic 160 160
lactic acid dissolves the enamel surface and create lesions within the 140 140 ***
Result 1: ***
enamel matrix, leading to tooth decay. 120 *** 120

Intensity

Intensity
Inoculate 5μL of monocultures and cocultures on phenol red BHI agar 5-8% CO2 is the ideal condition for coculturing the two 100 100 ***
While treatments for caries are available, they are often costly to bacteria species as it is the most physiologically 80 80
maintain, making them inaccessible to low-income families and for
plates and incubate in aerobic, 5-8 % CO2, microaerophilic, and 60 60
anaerobic in order to identify ideal coculturing condition
CO2 relevant and supports the growth of both species. 40 40
developing countries (the cost of maintaining a filling for a single lesion
accumulates to over 2000 dollars over a lifetime). In developing Figure 1a (left): Image of dental caries on molars
(Figure 4) 20 20
0 0
countries, over 90% of caries cases are not treated; even in developed Figure 1b (right): SEM Image of S. mutans PS1/Sm140 PS2/Sm140 Sm 140 PS1/Sm159 PS2/Sm159 Sm 159
countries, such as the U.K., dental loss caused by untreated caries (Ruiz-Linares 2014)
exceeds over 40% in prevalence. Inoculate monocultures and cocultures of the protective species and Microaerophilic Result 2: Figure 10: Acid intensity comparisons between ATCC S. mutans 140 Figure 11: Acid intensity comparisons between ATCC S. mutans 159
monocultures and cocultures (with protective species, PS1 and PS2). monocultures and cocultures (with protective species, PS1 and PS2).
6 different strains of S. mutans (2 from ATCC standards, 2 from caries- This protective species inhibits acid production of S.
Exposure to caries can have severe long-term effects, regardless of
whether a patient has had dental restoration. Due to fundamental free patients, 2 from caries-affected patients) mutans without hindering its growth under CO2 Figure 10 indicates that both cocultures of protective species and S. mutans have a significantly lower intensity of green in
changes within the oral microbiome that result from caries, patients with conditions, regardless of the strain of S. mutans used. comparison to the S. mutans monocultures for both S. mutans species (𝑝 < 0.001 in both coculture pairs of protective
previous exposure to caries are at a higher risk of severe periodontitis, species). Similarly, Figure 11 indicates that the protective species have significantly lower intensities of green in comparison
(Figure 5) to the monocultures (𝑝 < 0.001 in both coculture of protective species). Thus, based on the RGB color model, the lower
oral cancers, and heart disease. Because of the long-term ineffectiveness Anaerobic
of dental fillings and the inaccessibility of caries treatment, the current intensity of green correlates to a lower intensity of yellow, which translates to a lower intensity and production of acid.
Incubate plates for 48 hours at 5-8% CO2 and image plates under
movement in the dental community is to prioritize preventative
treatments over restoration. Despite this, current preventative standardized lighting with one light source to observe inhibitory effect Microelectrode Analysis
S. mutans from S. mutans from
measures, which have not been developed since the 1970s, are obsolete ATCC Standards caries-free patients caries-affected patients
as they do not specifically target the caries development process and are pH in S. mutans 140 pH in S. mutans 159
no longer effective with the rise of fluoride resistant S. mutans strains. Sm140 Sm159 Sm11-P1 Sm15-P1 Sm25-P1 Sm26-P1 Cultures and Cocultures Culture and Cocultures
Figure 2: Relative abundance of S. mutans between 8 ** ** 9
One emerging method of prevention is to harness the oral microbiome caries-free and caries-affected (S-ECC) subjects. Figure 5: Representative 8 **
7 **
to inhibit the mechanism behind caries development, which is the (Agnello et al. 2017) cultures of S. mutans and 7
Incubate starting cultures of S. mutans (ATCC standards) and cocultures of S. mutans and
6
6
production of lactic acid in S. mutans. With over 700 species of bacteria, 5
protective species for 48 hours protective species after 48- 5

pH

pH
the oral microbiome is an extremely diverse ecosystem of microbes. The 4
4
basis of modern oral microbiology research has been to understand hour incubation in 5-8% CO2 3 3
conditions. The yellow 2 2
these bacterial relationships and their overall impact on oral health. 6
1 1
regions are indicative of acid
Relative Abundance (%)

5 0 0
In the case of caries, the oral microbiome plays a very large role in production. PS1/Sm140 PS2/140 Sm140 PS1/Sm159 PS2/159 Sm159
determining not only susceptibility to caries but also susceptibility 4 Inoculate monocultures and cocultures of S. mutans and
against caries. A comparative analysis of the oral microbiome in caries- 3 protective species in the 24-well plate of phenol-red BHI agar Figure 12: pH comparisons between monocultures of S. mutans 140 Figure 13: pH comparisons between monocultures of S. mutans 159
free and caries-affected patients suggests that 1) caries susceptibility is 2 and cocultures of S. mutans with protective species. and cocultures of S. mutans with protective species.
not necessarily determined by S. mutans abundance and 2) there was an
1
upregulation of a particular species in the caries-free group, suggesting
protective properties – serving as the basis for this study [Figure 2, 3]. 0 Figure 12 demonstrates that the pH of the cocultures in both PS1 and PS2 with S. mutans 140 is significantly higher (and
Caries-Free Caries-Affected After incubating plate for 48 hours in 5-8% CO2, image the thus, less acidic) than that of S. mutans monoculture (𝑝 < 0.01 in both coculture of protective species). Furthermore, Figure
While the 20thcentury medicine saw progress in the decline of caries, 13 yielded very similar results with the pH of the cocultures being significantly higher and less acidic than its monoculture
plates in standardized lighting with one light source counterpart (𝑝 < 0.01 in both coculture of protective species). These results offer a clear demonstration that the
billions around the world still suffer from its long-term health and PS1 PS2 – control PS1 PS2 – control
Figure 3: Relative abundance of “Protective Species”
economic implications due to the cost and ineffectiveness of its genus in comparison between caries-free patients and
protective species does indeed prevent S. mutans from producing acid.
treatments. Thus, a new method to prevent caries is more important caries-affected patients. (Agnello et al. 2017)
than ever as it is still the most prevalent infectious illness without
effective preventative methods or treatments. Determine the acid intensity (specifically the intensity of
green in the RGB color model) of each culture in the PS1/Sm140 PS2/Sm140 Sm140
PS1/ PS2/ Sm140
Sm140 Sm140
phenol red plates through ImageJ software

PS1/ PS2/ Sm159

PS1/Sm159 PS2/Sm159 Sm159 The process of demonstrating and confirming the role of potential protective species in inhibiting acid production in S.
Sm159 Sm159
Inoculate monocultures and cocultures of S. mutans and mutans involved three stages: 1) preliminary coculturing, 2) colorimetry assays, and 3) microelectrode testing. In the first
stage, the study identified 5-8% CO2 as the ideal condition for coculturing because it was the only condition that satisfied the
protective species in a 24-well plate of phenol-red BHI agar three parameters necessary for successful cocultures: 1) the development of colones (demonstrating sufficient bacterial
growth, 2) the retention of traditional metabolic processes and 3) physiological relevance. In the second stage, the study
provided both qualitative and quantitative evidence of the protective species inhibiting acid production in S. mutans.
The study seeks to expand upon an observation made by comparative analysis behind caries-free and caries- Figure 6: 24-well plate images of monocultures Figure 7: RGB (Red, Green, Blue) colorimetric analysis of the 24- Qualitatively, within the phenol red plates, the area and intensity of yellow (indicative of acid production) decreases with the
and cocultures of ATCC Standards of S. mutans well plates – specifically the green value as it is an indicator of presence of protective species, regardless of the strain of S. mutans used, suggesting not only that protective species
affective patients in the upregulation of certain bacterial species in caries-free species [Figure 3]. The primary and protective species acid intensity. (See “Analysis” for statistical testing)
objective for this study is to identify and elucidate the properties of potentially “protective” bacterial species in
Calibrate the microelectrode with a standard of pH 4.0 prevent acid production in S. mutans but that it does so universally, regardless of the source/strain of S. mutans used. In
order to confirm this qualitative observation, the colorimetric assays using ImageJ and the microelectrode testing have
inhibiting the caries-causing mechanisms in S. mutans. In order to achieve this objective, the specific aims are: and another standard of pH 7.0 shown that protective species significantly decrease the acid production of S. mutans. Using these three methods, the
Sample pH Sample pH Sample pH hypothesis that protective species inhibits lactic acid production in S. mutans was supported.
Elucidate the interactions, particularly in relation to acid production, between S. mutans PS1 7.36 PS1 7.51 PS1 6.76
The final stage of experimentation, which involved testing the possibility of biofilm development of protective species,
and protective species in vitro PS2 7.45 PS2 7.38 PS2 7.40 yielded positive results as the SEM images clearly indicate that bacterial biofilm formation using the artificial saliva media
Measure the pH of the agar for each monoculture/coculture in the 24- PS1/Sm140 7.12 PS1/Sm140 7.50 PS1/Sm140 6.89 had been successful. Thus, in addition to demonstrating that protective species inhibits lactic acid production in S. mutans,
Determine the feasibility of biofilm development of S. mutans and protective species ex vivo well plate with a microelectrode PS1/Sm159 7.35 PS1/Sm159 7.48 PS1/Sm159 7.00 this study found that the protective species have the possibility of developing biofilms on teeth – a property of these
protective species that had not yet been previously observed.
PS2/Sm140 7.47 PS2/Sm140 7.17 PS2/Sm140 7.29
PS2/Sm159 7.60 PS2/Sm159 7.47 PS2/Sm159 7.11 These results open the doors to various future experimentation and applications. In the research setting, the discovery of
Sm140 5.45 Sm140 5.50 Sm140 5.51 biofilm feasibility in protective species will allow for ex vivo studies that measure the possibility of a reduction in enamel
dissolution through protective species. Further experimentation, such as gene expression assays and mass spectrometry,
Section third-molars from healthy individuals into four Sm159 5.44 Sm159 5.38 Sm159 5.45 allow for a deeper understanding of the mechanisms behind acid inhibition on a molecular level. In the clinical setting, the
sections using an IsoMet Precision Cutter acid inhibition by protective species could be developed into a preventative, probiotic drug that specifically targets the
Figure 8: pH of the agar, measured by a microelectrode, in 24-well plates of monocultures and ability of S. mutans to produce acid, leading to more effective preventative measures for caries.
cocultures of the protective species and S. mutans. (See “Analysis for statistical testing)

Create a specific area of the enamel in the third-molar Negative Control

A B E
section to allow for biofilm development and cover the
rest of the section (including the dentin and pulp) PS1
The past literature that analyzed the differences between caries-free and caries-affected bacteria
clearly found that an increased prevalence in bacterial species in the caries-free group, which, like
the caries-affected group, had very high amounts of S. mutans [Figures 2,3]. It is hypothesized that Submerge tooth in artificial saliva media and inoculate
the protective species (PS) would have an inhibitory effect on the acid production of S. mutans. bacteria into each respective well; incubate for 48 hours in
The inhibitory effect could be further explained by the diffusion of molecules by the protective 5-8% CO2 PS2 Agnello, M., Marques, J., Cen, L., Mittermuller, B., Huang, A., Chaichanasakul Tran, N., … Schroth, R. J. (2017). Microbiome Associated with
Severe Caries in Canadian First Nations Children. Journal of Dental Research, 96(12), 1378–1385. https://doi.org/10.1177/0022034517718819
species that inhibit the gene expression within the glycolytic pathways in S. mutans, thereby
hindering acid from being produced. Ajdic, D., McShan, W. M., McLaughlin, R. E., Savic, G., Chang, J., Carson, M. B., … Ferretti, J. J. (2002). Genome sequence of Streptococcus

x x
mutans UA159, a cariogenic dental pathogen. Proceedings of the National Academy of Sciences, 99(22), 14434–14439.
Fix teeth in iron formalin for 24 hours and dehydrate with C D Result 3: https://doi.org/10.1073/pnas.172501299
increasing concentrations of ethanol in 30-minute intervals; In the ex vivo model, all S. mutans
C3H6O3 C3H6O3 Dewhirst, F. E., Chen, T., Izard, J., Paster, B. J., Tanner, A. C. R., Yu, W.-H., … Wade, W. G. (2010). The Human Oral Microbiome. Journal of
C3H6O3 C3H6O3 sputter coat with platinum and this protective species can
Bacteriology, 192(19), 5002–5017. https://doi.org/10.1128/jb.00542-10
develop biofilms on teeth under
artificial saliva media. Featherstone, J. (2004). Caries Management by Risk Assessment: The Caries Balance. San Francisco, CA: University of California San Francisco.
Sm PS Sm

C3H6O3
C3H6O3

Visual Representation of the Proposed Acid Inhibition Process

x x
C3H6O3
C3H6O3
Perform SEM microscopy to observe biofilm development at
75,000 times magnification
Sm140 Sm159 Figure 9:
SEM images of a) PS1, b) PS2, c) Sm140, d)
Sm159 and e) negative control after being
incubated on teeth for 48 hours in artificial
saliva under CO2 conditions
Featherstone, JDB. (2008). Dental caries: a dynamic disease process. Australian Dental Journal, 53(3), 286–291.
https://doi.org/10.1111/j.1834-7819.2008.00064.x

Krishnan, K., Chen, T., & Paster, B. (2016). A practical guide to the oral microbiome and its relation to health and disease. Oral Diseases, 23(3),
276–286. https://doi.org/10.1111/odi.12509

All images, charts or graphs were created by the finalist except as noted.