Investigation into Compound(s) Exhibiting Streptococcus mutans Anti-biofilm Activity This thesis is submitted by SUPANAN SUCHARIT For the degree of Doctor of Philosophy (PhD) In the Faculty of Medical Sciences University College London Department of Microbial Diseases UCL Eastman Dental Institute 256 Gray’s Inn Road London WC1X8LD, UK -2020- Declaration I, Supanan Sucharit, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. A metagenomic library used in this study was constructed by Dr Liam Reynolds (Eastman Dental Institute, UCL). An environmental bacterial isolate library was isolated by Dr Liam Reynolds under Swab and Send project. i | P a g e Abstract Streptococcus mutans is one of the main oral bacteria causing caries through biofilm formation. Bioactive compounds, derived from microorganisms, that disrupt S. mutans biofilms are a potential treatment for dental caries. A high throughput microtitre plate biofilm assay was developed to screen an oral metagenomic library and an environmental isolate library for S. mutans clones that produced anti-biofilm compounds. Anti-biofilm activity was not found in 6,624 screened metagenomic clones. Two different kinds of anti- biofilm activity, biofilm detaching activity and biofilm inhibitory activity, were found to be produced by seven isolates from the environmental library. The biofilm detaching activity was produced by all seven isolates, which were identified as Bacillus velezensis based on whole genome sequencing results. Isolate 6E produced the compound with the biofilm detaching activity in M9 minimal medium supplemented with sucrose. The activity disappeared after boiling and treatment with proteinase K, suggesting that the compound(s) responsible for the biofilm detaching activity was a protein. It was hypothesized that the activity might be due to levansucrase, which produce levan, however commercial levan had no effect on S. mutans biofilm formation. The biofilm detaching activity was not effective when S. mutans biofilms were allowed to form on saliva-coated surfaces. This suggests that the biofilm detaching activity might not be applicable for dental caries protection. The biofilm inhibitory activity was produced by three isolates: 6E, 10G, and 11E. The biofilm inhibitory activity was not detected when the supernatant was ii | P a g e boiled. Many purification methods were used to separate the biofilm inhibitory however the compound has not yet been purified The compound derived from B. velezensis 6E, 10G, 11E. with S. mutans biofilm inhibitory activity in this study may have potential to be applied as an anti-dental caries agent and should be investigated further in the future. iii | P a g e Impact statement Dental caries is one of the most common oral diseases which can lead to lifelong effects. S. mutans is one of the main oral bacteria causing caries through adhesion, acid and biofilm formation. Research on identifying a way to inhibit biofilms produced by this major cariogenic pathogen might potentially lead to a means for the prevention and treatment of dental caries. Therefore, this study focused on identifying potential compounds that could inhibit biofilm formation by S. mutans. The results presented in this thesis have identified new biological tools to target S. mutans biofilms. Two anti-biofilm activities were identified in novel strains of Bacillus velezensis, isolated from environmental samples in UK. The first anti- biofilm activity was a biofilm detaching activity that was detected in supernatants derived from seven strains of B. velezensis. The supernatants allowed S. mutans to grow and form biofilms but they caused the biofilms to easily detached. However, the biofilm detaching activity was not effective when S. mutans biofilms were allowed to form on saliva-coated surfaces. This finding provides knowledge for targeting S. mutans biofilm formation and paves the way for future studies to fully elucidate the mechanism of this biofilm detaching activity. The second anti-biofilm activity detected in this study was a biofilm formation inhibitory activity. The supernatants derived from three strains of B. velezensis could inhibit biofilm formation from S. mutans on saliva-coated surfaces. The compound responsible for the biofilm formation inhibitory activity was subject to a number of purification methods but the compound has not been purified. iv | P a g e This compound may have the potential to be used as an agent for the prevention of dental caries and further attempts should be made to isolate the agent to allow further investigation in the future. Currently dental caries is still widespread, even in developed countries, and compounds that could prevent or reduce S. mutans biofilms would be of huge benefit. The work presented in this thesis has identified bacteria that produce molecules that can inhibit S. mutans biofilm formation and which may lead to the development of novel treatment for dental caries. v | P a g e Acknowledgement Since, three years working together, I can say that this a period of three years in the lab can change my life especially my attitude in a more scientific way. Thank you very much to Dr Sean Nair, you are the great supervisor and you can be a model of a good teacher that I can follow up. Dr Adam Robert, thank you very much for giving me an opportunity and always support particularly in my difficult situations of my PhD. Without all of you, I could not finish my PhD. I also would like to acknowledge the help of my colleagues and staffs in Department of Microbial Diseases, UCL Eastman Dental Institute, who gave the suggestions during the lab work and permission to use all equipment and material to complete this project. Mr Liam J. Reynolds, UCL Eastman Dental Institute, isolated environmental bacteria from Swab and Send project. Dr Haitham, who is my early morning lab buddy, Dr Anna, Tracy, Ingrid, Mehmet, Marika, Catie, Deena, Hadeel, Ahmed, Shata, Khadija, Enas, Ajijur, Khalid. Especially, Supathep, Asyura, Sophia, Shirene, Arely, Zeina, Ladan and Erni. Thank you Thai PhD students for your companions: Thanapong, Chaiyong, and Chutipong, Akkapon, Nat, Papon, Theerasak, Suthathep, Chatdanai, Weeranan, Kanokwan and more, MWIT teachers: O-phart, Thanakrit, Sarot, Nitat and Sittichock. My colleagues in MWIT especially Biology department, particularly Sataporn, Sirimad and Tippanart. My best friend forever, Kamonrat and her friends in Birmingham, Surasak, Shane and Cherngchai. I then would like to thank my sponsor both Royal Thai Government and MWIT to provide an opportunity to me for pursuing my PhD. Finally, I would like to thank my friends and my previous students. Particularly, I would like to express my gratitude to my family, my parents and my siblings (Kea, Kuk, Kof). As a mum doing PhD, I could not finish this thesis without my great inspiration, thank you very much Nitchanan (Goonny) for giving me energy and everything. You are the answer who I live for. The last but not least, my beloved husband, Yutapon Sucharit (Bee), for his always support and dedicates himself to my PhD. Great thanks for always beside me in many difficult situations. Thank you for your love. vi | P a g e Table of Contents General introduction ...................................................... 2 Dental Caries .......................................................................... 2 Cariogenic oral bacteria .......................................................... 8 Streptococcus mutans............................................................11 General information ...................................................................11 Virulence factors........................................................................13 Oral biofilm .............................................................................22 Composition ..............................................................................22 Biofilm formation........................................................................23 Biofilm dispersion ......................................................................25 Possible strategies to target S. mutans biofilm ......................27 Inhibiting the initial adhesion of S. mutans ................................27 Targeting EPS ...........................................................................30 Direct interfering the survival of S. mutans biofilm cells ............31 Induction of biofilm dispersal .....................................................33 Bacillus spp. ...........................................................................34 General information ...................................................................34 A source of bioactive compounds..............................................35 Aim and objectives .................................................................42 Materials and Methods ..................................................44 Bacterial strains .....................................................................44 S. mutans biofilm formation ....................................................44 Crystal violet quantification of biofilm formation .....................44 vii | P a g e Screening for anti-biofilm activity from an environmental isolate library. 45 Primary screening .....................................................................45 Secondary screening .................................................................46 Heat stability