Culture-independent analysis of the bacteriology associated with acute otitis media in Indigenous Australian children. Robyn Marsh BAppSc (MLS), MSc Thesis submitted for the degree of Doctor of Philosophy December 2011 Child Health Division Menzies School of Health Research Charles Darwin University Darwin, Australia ii Declaration I hereby declare that the work herein, now submitted as a thesis for the degree of Doctor of Philosophy of the Charles Darwin University, is the result of my own investigations, and all references to the ideas and work of other researchers have been specifically acknowledged. I hereby certify that the work embodied in this thesis has not already been accepted in substance for any degree, and is not being currently submitted in candidature for any other degree. Robyn Marsh December 2011 iii Abstract Otitis media is endemic in many remote Indigenous Australian communities. Children in remote communities develop otitis media in the first weeks of life and are at high-risk of progression to chronic suppurative otitis media (CSOM). Current therapeutic and preventive interventions are of limited benefit. This thesis presents a culture-independent analysis of the bacteriology associated with acute otitis media in Indigenous children from the Northern Territory. The objective of the study was to use culture-independent methods to better understand the bacteriology underlying acute otitis media in this population. Principle findings from the study are as follows: 1. Nasopharyngeal total or pathogenic bacterial loads are unsuitable as prognostic indicators of clinical antibiotic treatment outcomes in Indigenous children with acute otitis media. 2. Alloiococcus otitidis is present in ear discharge from Indigenous children with acute otitis media, potentially as a secondary middle ear pathogen. Further studies to test for A. otitidis in CSOM are warranted. 3. T-RFLP can provide broad characterisation of bacterial communities in upper respiratory specimens; however, it is limited by methodological biases which result in underestimation of bacterial richness. 4. Despite the methodological limitations, T-RFLP analysis demonstrated significant differences between nasopharyngeal and ear discharge bacterial communities in Indigenous children with acute otitis media with perforation, suggesting divergence of the middle ear microbiome following secondary infection by canal flora. 5. 16S rRNA gene microarray (PhyloChipTM) analysis of ear discharge from Indigenous children with acute otitis media with perforation indicates a high- level of bacterial richness which is hypothesised to contribute to progression to CSOM. iv Prospective longitudinal studies are now required to better understand the canal flora before perforation, the microbiology of acute otitis media immediately following perforation, and microbiomic changes associated with prolonged perforation. Such studies will aid understanding of the pathogenesis of acute otitis media with perforation and progression to CSOM, and potentially reveal new therapeutic targets or prevention strategies. v Acknowledgements This study contributes a small part to the wider Ear Health research program at Menzies School of Health Research. I am indebted to Amanda Leach and Peter Morris, the Ear Team Leaders, for their advice and ongoing support. Thank you both for always reminding me of the broader context of the research. Your continued efforts to achieve meaningful improvements in ear health for Indigenous children are inspirational. Amanda, I’m especially thankful to you for helping me see that it was possible to do a PhD while raising a toddler. I am also indebted to the Indigenous communities and families that have supported, and continue to support, ear health research projects. In particular, I would like to thank the Indigenous communities and families that participated in the AATAAC study who provided the specimens used in my analyses. It is my sincere hope that this work will in some way contribute to improved health for your children. I was blessed to have had Heidi Smith-Vaughan as my supervisor. Heidi, your enthusiasm for all things microbiological is infectious (pun intended), and was instrumental in my decision to commence doctoral studies. Your endless patience, wisdom and assistance during my study are deeply appreciated. I feel privileged to have travelled this path under your guidance. Thank you for everything you have made possible, for your friendship, encouragement, vision and for always making time for me. Most especially thank you for coming to Seattle – it wouldn’t have been the same without you! Although not a formal supervisor, Mirjam Kaestli continually provided guidance in times of need. Thank you, Mirjam, for all your insight, advice and enthusiasm. I am particularly grateful for your assistance with statistical analyses and hope some day to repay the many favours. I would also like to thank Allen Cheng and Linda Ward for their assistance with statistical analyses. Allen, you are amazing! Thank you for all your help and for commenting so insightfully on my drafts. Linda, thank you for always being ready with an enthusiastic mind, and teaching me that evolution is better than revolution! I’m also indebted to Anne Chang for her encouragement and support. Thank you, Anne, for your advice, support and vision. vi Enormous thanks also go to all the Menzies lab staff. Thank you to everyone who has endured my random chats, rants and brainstorming. In particular, thank you everyone in the Child Health Lab group. You are a fantastic team and I feel lucky to have conducted my study alongside such a great group of people. Michael Binks, thanks for always being there to provide feedback and bounce ideas off – it’s appreciated more than you know. Susan Pizzutto, thanks for always listening and understanding when I just needed to purge. Thanks also to everyone who helped with laboratory analyses, in particular Jemima Beissbarth and Peter Christensen for their help processing PCRs and T-RFLPs, and Kim Hare and Vanya Hampton for their help with culture-based analyses. I’d also like to thank Asha Haslem for teaching me to drive the GeneAnalyzer; Tegan Harris, Rachel Lilliebridge and Leisha Richardson for their assistance sourcing reference strains; and Jo Bex for her assistance with all things logistical. Thanks also to the wider Menzies staff that have helped along the way. Gabby McCallum, thank you for so willingly providing help whenever I came looking for more information. Robyn Liddle, thank you for always having an answer whenever I needed to drag something out of a database. Maria Scarlett, your advice and assistance with all things ethical is much appreciated. Martin Klein and Trevor Hopps, thanks for coming to the rescue whenever I needed IT assistance. Thanks also to Kalotina Halkitis, Jill Albion, Joan Kieboom, Kate McKay and Jess Singleton for all your administrative assistance. Special thanks to my office buddies, Wajahat Mahmood, Jacklyn Ng and Yuwana Podin for their support, advice, chats and the shared chocolate moments during my final write-up. I’m also grateful to Lucas Hoffman (University of Washington) for his advice during preparation for microbiomic analyses, critical comments on grant applications and his contribution to my caffeine addiction. Thanks for always making time for me, Luke. Thanks also to Geraint Rogers (King’s College London) and Susan Lynch (University of California, San Francisco) for their advice regarding DNA extraction for microbiomic analyses. vii Deepest thanks go to my family. Andy Hansen, thank you for so enthusiastically responding when I first started talking about taking on a PhD. You’ve endured all my ups and downs with your trademark patience. Thank you for helping me see this through to its end and for making it possible for me to pursue my dreams. Zach Leto, while it may not mean much to you now, I am eternally grateful for your cuddles and smiles and for always reminding me that there IS more to life than my thesis. Judy and Barry Hansen, thank you SO much for all the hours of childcare and for keeping me nourished whenever things became too busy. Thanks also to Gail Marsh, Christopher Marsh, Cassandra Marsh and Mavis Watson for their support and for all the parcels they sent. Ray Marsh, thank you for coming to the rescue when Andy was out bush. This project was made possible by funding from the National Health and Medical Research Council, the Channel 7 Children’s Research Foundation, the Trust Foundation and Menzies School of Health Research Ochre Funds. I thank these organisations for their support. I also would like to thank the Future Leaders Foundation for supporting my travels to the United States during 2011. viii Publications and presentations This section describes publications and presentations made during my PhD candidature. PUBLICATIONS Publications in refereed journals Marsh R, Smith-Vaughan H, Hare KM, Binks M, Kong F, Warning J, Gilbert GL, Morris P, Leach AJ. 2010. The nonserotypeable pneumococcus: phenotypic dynamics in the era of anticapsular vaccines. J Clin Microbiol. 48(3):831-5. (Equal first authorship) Marsh R, Smith-Vaughan H, Mackenzie G, Fisher J, Morris PS, Hare K, McCallum G, Binks M, Murphy D, Lum G, Cook H, Krause V, Jacups S, Leach AJ. 2009. Age- specific cluster of cases of serotype 1 Streptococcus pneumoniae carriage in remote Indigenous communities in Australia. Clin Vaccine Immunol.16(2):218-21. (Equal first authorship) Conference proceedings Kirkham LA, Wiertsema SP, Smith-Vaughan HC, Thornton RB, Marsh RL, Lehmann D, Leach AJ, Morris PS, Richmond PC. 2010. Are you listening? The inaugural Australian Otitis Media (OMOZ) workshop-towards a better understanding of otitis media. Med J Aust. 193(10):569-71. Invited reviews Smith-Vaughan H, Marsh RL, Leach AJ. 2009. Otitis media: an ongoing microbial challenge. Microbiology Australia. 30(5):181-4. Submitted for publication Binks, MJ, Temple, B; Kirkham, L; Wiertsema, S; Dunne, E; Richmond, P; Marsh, RL; Leach, AJ; Smith-Vaughan, HC. Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays.