Effects of Neisseria sp. on the Oral  Biofilm Formation of Streptococcus mutans

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Effects of Neisseria sp. on the Oral Biofilm Formation of Streptococcus mutans
James, Anika
Smith, Amanda
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Denver, CO
Metropolitan State University of Denver
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Collected for Auraria Institutional Repository by the Self-Submittal tool. Submitted by Matthew Mariner.
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Faculty mentor: Sheryl Zajdowicz
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Major: Biology

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Effects of Neisseria sp. on the Oral Biofilm Formation of Streptococcus mutans Anika James, Amanda Smith, and Sheryl Zajdowicz , Ph.D. Department of Biology, Metropolitan State University of Denver, CO The human oral microbiome houses a wide variety of microbes, both pathogenic and commensal . Streptococcus mutans , due to its acidogenic nature and ability to form biofilms, is a primary cause of dental caries, gum inflammation, and periodontal disease in humans 1 , 2 . Various Neisseria sp . exist as commensal species in the human oral cavity and nasopharynx 4 . While research has begun to elucidate the complex interplays between members of the oral microbiome, limited information exists regarding the interactions between Neisseria sp . and S . mutans . Previous studies on Neisseria sp . demonstrated its ability to catabolize lactate, including that produced by S . mutans . Therefore, this research aims to explore the effect Neisseria sp . have on the ability of S . mutans to form a biofilm . To study this effect, mature biofilms were formed for each organism and the effect of Neisseria sp . as a pioneer colonizer versus a secondary colonizer on S . mutans biofilm formation is under investigation . Strategies to promote commensal growth and to limit S . mutans are also being evaluated, which may provide insight on ways to promote oral health . Abstract Optimum growth conditions for S. mutans and N.sicca were determined by growing them in BHI broth, with 5% CO 2 , and a The appropriate cell concentrations and conditions were determined for growing these organisms in a bi species biofilm in 1% sucrose+BHI 2 , S. mutans biofilms were grown on composite resin disc like surfaces with BHI broth+ 1% sucrose in a 24 well plate for 48 hours (Chart 1 outlining steps carried out to form a biofilm). Following formation of mature S. mutans biofilms, the discs were transferred to fresh wells with varying amounts of N. sicca. Positive controls were performed for S. mutans , and for high and low N. sicca abundance. After 48 hours, the biofilms were disrupted and colony forming units (CFUs) for both organisms in the experimental and control groups was determined.(Fig 5 and 6 ). Since no significant statistical differences were observed in cell counts, the growth conditions and balance of cell concentrations need to be reevaluated . Observations of plated dilutions indicate that intact S . mutans might be producing something inhibitory that blocks the growth of N . sicca when grown together . This shows that trying to add N . sicca to disrupt an already existing S . mutans biofilm is not a viable option for control . A future experiment would be to form N . sicca biofilm first and then add S . mutans at varying cell numbers . Growth will also be tested under varying concentrations of sucrose and varying cell number concentrations . Additionally, evaluation of the effect of other Neisseria sp . ( e . g N . lactamica ) and other commensal Streptococcus sp . on mature S . mutans biofilms could be carried out . Utilization of other biofilm quantification methods will be carried out in the future . Streptococcus mutans is found in the human oral cavity and is a contributor to tooth decay 1 , 2 , which affects almost 3 . 9 billion people worldwide with dental caries and other periodontal diseases 6 . This prevalence suggests a need for improved understanding of the contributing role of pathogenic microbes to dental diseases . Several Streptococcus sp . exist in the oral cavity and utilize sucrose and other dietary sugars which are then converted to lactate under aerobic conditions 3 . This lactate further converts to volatile acids that lead to the forming of lesions on the surface of teeth 3 . I would like to thank my research mentor, Dr . Sheryl Zajdowicz , for her constant support and guidance for this project . I also acknowledge, Amanda Smith for defining this project, prior to my arrival in the lab . Further, I would like to thank Lisa Gotow and Mark Karlok from the Biology department at MSU Denver, for their helpful advice . Financial support for this research was provided by MSU Department of Biology, MSU Undergraduate Research Program and CO WY AMP Research Grants . Acknowledgements Figure 2: Streptococcus mutans Figure 1: Electron microscope images of 16 hour old S. mutans biofilm 7 Lactate degradation in the oral cavity is therefore investigated in this project by introducing Neisseria sp . such as N . lactamica and N . sicca which are known to metabolize lactate 3 . Both N . sicca and N . lactamica are Gram negative diplococci, that colonize the oral cavity and human nasopharynx 4 . We propose that Neisseria sp . will disrupt biofilm formation by S . mutans . Chart 1 : Formation of mature biofilms of S. mutans and N. sicca Figure 4: Experimental plate after 48h growth. Well 1 is the positive control for S. mutans . Wells 2 and 3 are the low (OD 600 =0.1 and high (OD 600 =0.25) amounts of N. sicca controls, respectively. Wells 4 and 5 are mature S. mutans biofilm discs with low and high (0.25) cell abundance of N. sicca , respectively. Figure 6 : Experimental and Positive Control BHI agar plates a) S. mutans biofilm dilutions (positive control) b) N. sicca low concentration biolfilm dilutions c) N. sicca high concentration biofilm dilutions d) Mature S. mutans biolfilm with low concentration N. sicca e)Mature S. mutans biofilm with high concentration N. sicca S. mutans and Neisseria sp. Biofilm Formation 1)Prepared bacterial suspension in 5ml saline 2) Measured OD 600 of culture/suspension 3) Aseptically transferred discs to wells 4) Transferred 1 ml of culture at OD 600 =0.1 or 0.05 to wells in microtiter plate hours. 6) Determined cfu / mL. Successful growth was observed for both experimental and control plated dilutions. Biofilm formation was observed in the wells where S. mutans was grown. After growing N. sicca (both high and low concentration) with mature S. mutans biofilm, no N. sicca colonies were observed on the plates with S. mutans . S. mutans colonies reduced in numbers (Table 1), when a higher concentration of N. sicca was introduced, compared to S. mutans plated dilutions. A statistical difference in cell counts, was not observed, which were all calculated to be 10 6 on average. Culture CFU/mL recovered from Biofilm S. mutans ( OD 600 =0.1) 1.45x10 7 N. sicca (low concentration, OD 600 =0.1) 2.09x10 6 N. sicca (high concentration, OD 600 = 0.25) 3.6x10 6 Mature S. mutans Biofilm + N.sicca low concentration 9.2x10 6 Mature S. mutans Biofilm + N.sicca high concentration 2.45x10 6 Table 1: Bacterial CFU/mL recovered from Mature Biofilms of S. mutans and N. sicca Figure 3: Neisseria sicca Email: 1. Diaz, P. I., et al. (2006). Molecular Characteriziation of Subject Specific Oral Microflora during Initial Colonization of Enamel. Applied and Environmental Microbiology , 2837 2848. 2. Matsumoto Nakano, M., et al. (2017). Role of Streptococcus mutans surface proteins for biofilm formation. Okayama, Japan: Japanese Dental Science Review . 3. Hoshino, E., et al. (1976). Lactate degradation by a strain of Neisseria isolated from human dental plaque. Archives of Oral Biology (21) 11:677 683. doi 10. 1016/0003 9969(76)90142 4 4. Pandey A.K., et al. (2017) Neisseria lactamica Y92 1009 complete genome sequence. Standards in Genomic Sciences , 12:41 DOI 10.1186/s40793 017 0250 6 5. Berger, D., et al. (2018). Oral Biofilms: Development, Control, and Analysis. MDPI , 7(3): 24. 6. World Dental Federation. ( n.d. ). Fact, Figures and Stats . Retrieved from Oral disease: 10 key facts: 7. ( n.d. ). Streptococcus mutans biofilm. Retrieved from Microbe Wiki: 8. Waters, M. S., et al. (2014) Microstructure and Mechanical Properties of In Situ Streptococcus mutans Biofilms ACS Appl. Mater, 327 332 9. Tagaino , R., et al. (2019) Metabolic property of acetaldehyde production from ethanol and glucose by oral Streptococcus and Neisseria . Scientific Reports, 9: 10446 019 46790 9 References Figure 5: Disrupted biofilm is diluted and plated onto BHI agar, for about 48 hours in a 5% CO 2. incubator Introduction Methodology Results Conclusion and Future Directions