Effects of children’s toothpastes and mouth rinses on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus Alana Evans BDSc A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2014 School of Dentistry Abstract Objectives: As suppression of mutans streptococci in young children may prevent or delay colonisation of the oral cavity, tooth brushing with dentifrices containing anti- Streptococcus mutans activity may aid in preventing caries. The aims of this study were to compare the effects of children's toothpastes, mouth rinses, oral antiseptics and fluoride solutions on the growth of Streptococcus mutans and non-mutans bacteria (Streptococcus sanguinis and Lactobacillus acidophilus). Methods: The agar diffusion assay at neutral pH was used to determine bacterial growth inhibition. Zones of bacterial inhibition were measured using a micrometer gauge. Results: Dentifrices containing 1,450 ppm fluoride produced greater growth inhibition of both S. mutans and S. sanguinis than those with < 500 ppm. No inhibition was seen for pure solutions of sodium fluoride or sodium monofluorophosphate at fluoride concentrations up to 10%. Stannous fluoride exerted anti-bacterial effects at concentrations above 1%. Significant growth inhibition of both S. mutans and S. sanguinis was seen with sodium lauryl sulphate at 0.25% and with triclosan at 0.01%. The mouth rinses containing 2% chlorhexidine gluconate, 0.05% cetylpyridinium chloride and 0.05% sodium fluoride produced anti-bacterial effects against S. mutans, S. sanguinis and L. acidophilus. Of the pure compounds, 0.01% chlorhexidine produced the greatest zone of growth inhibition against S. mutans. Of the combinations tested, 0.1% sodium fluoride with 5% povidone iodine produced synergistic anti-bacterial effects against S. mutans and S. sanguinis. The combination of 10% povidone iodine with 0.5% sodium hypochlorite produced additive anti-bacterial effects against L. acidophilus. Interference was seen in some combinations such as chlorhexidine with sodium lauryl sulphate, most likely through anion-cation reactions. However, 0.1% sodium fluoride when combined with 0.01% chlorhexidine did not interfere with the anti-bacterial effects of chlorhexidine alone 2 against S. mutans or S. sanguinis, but it reduced the anti-bacterial effects of cetyl pyridinium chloride alone against these bacteria. Conclusion: Sodium lauryl sulphate and triclosan are major bacterial inhibitory compounds in children’s dentifrices. Mouth rinses containing chlorhexidine, sodium fluoride and cetyl pyridinium chloride have growth inhibitory effects against Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus. The combinations of povidone iodine with sodium hypochlorite and povidone iodine with sodium fluoride produced additive and synergistic effects respectively. 3 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 4 Publications during candidature (1) Inhibitory effects of children's toothpastes and fluoride compounds on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus - accepted for publication in 2014 (2) Effect of antiseptic mouth rinses on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus - accepted for publication in 2014 (3) Anti-bacterial effects of combinations of oral antiseptics and sodium fluoride on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus - submitted for publication in 2014 Publications included in this thesis (1) Inhibitory effects of children's toothpastes and fluoride compounds on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus - incorporated as Chapter 3 Contributor Statement of contribution Designed experiments (75%) Dr Alana Evans (Candidate) Wrote and edited the paper (75%) Dr Lei Chai Designed experiments (5%) Designed experiments (20%) Prof. W. Kim Seow Wrote and edited paper (20%) Prof Laurence Walsh Wrote and edited paper (5%) (2) Effect of antiseptic mouth rinses on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus - incorporated as Chapter 4 Contributor Statement of contribution Designed experiments (75%) Dr Alana Evans (Candidate) Wrote and edited the paper (75%) Dr Lei Chai Designed experiments (5%) Dr Shaneen Leishman Wrote and edited paper (5%) Prof. W. Kim Seow Designed experiments (20%) 5 Wrote and edited paper (15%) Prof Laurence Walsh Wrote and edited paper (5%) (3) Anti-bacterial effects of combinations of oral antiseptics and sodium fluoride on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus acidophilus - incorporated as Chapter 5 Contributor Statement of contribution Designed experiments (70%) Dr Alana Evans (Candidate) Wrote and edited the paper (75%) Designed experiments (5%) Dr Shaneen Leishman Wrote and edited paper (5%) Designed experiments (25%) Prof. W. Kim Seow Wrote and edited paper (15%) Prof Laurence Walsh Wrote and edited paper (5%) 6 Contributions by others to the thesis Professor Laurence Walsh: contributed in providing laboratory guidance to the research and in writing the publications. Dr Shaneen Leishman: contributed in providing laboratory guidance to the research, analysis of results and in writing the publications Dr Lei Chai: contributed in providing laboratory guidance to the research Statement of parts of the thesis submitted to qualify for the award of another degree None 7 Acknowledgements I would like to thank the many people who have contributed to this research and supported me throughout this project. Without their guidance, encouragement and smiles, this research would not have been possible. I would firstly like to say a huge thank you to Professor Kim Seow, my supervisor and mentor. Her absolute passion for research and expanding knowledge in the field of pediatric dentistry gave life to this research project. This also gave life to my own interest and passion for research and provided me with constant motivation. When the project was not going according to plan, Kim always had a level head and taught me how to reassess the work being done to reach the desired outcome. Her patience and willingness to help with all my questions throughout the laboratory work and writing as well as her organisation, kept me on track and made this project and my life run more smoothly. I am sincerely appreciative of all the time, care and dedication Kim has put into this project and to helping guide me through this research. It has been a privilege to work with her. Similarly, I would like to thank Professor Laurie Walsh as my associate supervisor. He provided much guidance of the research and aid in editing of the final papers. His friendly personality made it a delight to work with him and his positive emails helped to keep me on track. I would also like to say a huge thank you to Dr Lei Chai and Dr Shaneen Leishman for their assistance in the laboratory with both design and running of the experiments. Further, all the input Shaneen has provided in the analysis of results and writing of papers was invaluable. Simply seeing these beautiful ladies smiling in the laboratory made the process an enjoyable experience. Their interest in this project and care of how I was going was greatly appreciated. I would like to thank the University of Queensland School of Dentistry Research Committee: Miss Jodie Caruana, Mrs Andrea Kazoullis, Dr Shazia Naser-ud-Din, Dr Annetta Tsang, Dr Pauline Ford, and Dr Sowmya Shetty for the administrative 8 running of the postgraduate masters degree and for their advice and critique which helped to direct and mould this research and help me develop my research skills. Finally, I would like to thank my family and friends for their ongoing support and care throughout this degree. To my parents, Peter and Alex, thank you for providing a great home environment and escape from my daily stresses, for managing my moods so carefully and for your endless support and love. To my brother, Lachlan, thank you for the