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Cecilia Gonzales Marin

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Cecilia Gonzales Marin MOLECULAR DETECTION OF BACTERIA FROM A POSSIBLE MATERNAL ORAL ORIGIN IN NEONATAL GASTRIC ASPIRATES OBTAINED FROM COMPLICATED PREGNANCIES Thesis submitted to the University of London to obtain the degree of DOCTOR OF PHILOSOPHY Cecilia Gonzales Marin Institute of Dentistry Barts and The London School of Medicine and Dentistry Queen Mary, University of London 2011 SUPERVISORS: Rob Allaker, PhD Queen Mary University of London Barts and The London School of Medicine and Dentistry Centre for Clinical and Diagnostic Oral Sciences David Spratt, PhD University College London Eastman Dental Institute Division of Microbial Diseases 2 ABSTRACT It has been suggested that periodontal disease, a disease that affects the supporting tissues of the teeth, represents a risk factor for adverse pregnancy outcomes. Certain oral pathogens possess a demonstrated ability to translocate and invade the amniotic tissues. Once in the amniotic environment, these opportunistic colonisers could then initiate or contribute to a perinatal infection, and in this way be involved in the complications. The overall aim of this study was to determine the presence, and confirm the origin, of suspected maternal oral microbiota in neonatal gastric aspirates (swallowed amniotic fluid) collected due to complications during pregnancy and/or evidence of neonatal sepsis. Non-cultural PCR-based methods directed to the ribosomal encoding genes (rDNA) were applied to analyse neonatal and maternal samples. The use of universal and species-specific primers that target the bacterial 16S rRNA gene allowed identification and quantification of broad-range and specific bacteria to the species level. Sequence comparative analysis of a more variable fragment, the intergenic spacer region located between the 16S and the 23S rDNA, was finally used to compare strains obtained from the neonates and their counterparts in the respective mother’s oral and vaginal samples. Data analysis allowed identification of a range of potential confounding factors for presence of bacteria in the infants, such as vaginal delivery and prolonged rupture of membranes. However, bacteria with a possible oral origin were mostly identified in the neonatal samples in low prevalence and not associated with any particular variable. Quantitative analysis of potential periodontal pathogens demonstrated the presence of Porphyromonas gingivalis (1%) and Fusobacterium nucleatum (16%) in the neonates. F. nucleatum was detected at relatively high levels (mean=4.41E+02 cells/ml); representing up to 50% of the total bacterial load, which strongly supports its possible role in pregnancy complications. Also, F. nucleatum subspecies analysis and comparisons at the strain level suggest the oral cavity as the most likely origin of this infectious agent. This study supports the need for further studies. 3 Declaration I hereby certify that the work embodied in this thesis is the results of my own investigation, except where otherwise stated. 4 LIST OF PUBLICATIONS/PRESENTATIONS Publications Gonzales-Marin C, Spratt DA, Kempley S, Millar MR, Simmonds M, Allaker RP. Identification of bacteria in neonates at risk of infection delivered by caesarean and vaginal birth. [Manuscript submitted to the American Journal of Obstetrics and Gynaecology]. January 2011. Gonzales-Marin C, Spratt DA, Kempley S, Millar MR, Simmonds M, Allaker RP. Levels of periodontal pathogens in neonatal gastric aspirates and possible maternal sites of origin. [Manuscript submitted to the journal Molecular Oral Microbiology]. February 2011. Gonzales-Marin C, Spratt DA, Millar MR, Allaker RP. The 16S-23S rDNA intergenic spacer region to determine the precise origin of Fusobacterium nucleatum in neonates. [Manuscript submitted to the journal Molecular Oral Microbiology]. March 2011. Presentations Title: Identification of oral bacteria in neonatal gastric aspirates. Meeting details: Society for General Microbiology. Spring Meeting. Harrogate International Centre, North Yorkshire. 31 st March – 2nd April, 2009. Acknowledgement: Media release. Title: Analysis of the 16S-23S rDNA intergenic spacer region of F. nucleatum and G. elegans from a possible oral origin identified in neonatal gastric aspirates. Meeting details: Gordon Research Conference on Periodontal Disease. New Hampshire, USA. 2nd – 7th August, 2009. Prize awarded: GRC funding award for young investigators. 5 Title: Detection and quantification of periodontopathogens in neonatal gastric aspirates using quantitative PCR. Meeting details: British Society for Oral and Dental Research- IADR, Scientific Meeting. Glasgow, Scotland. 1st – 4th September, 2009. Prize awarded: Postgraduate funding award. Tittle: Molecular techniques to study microbiology of gastric aspirates. Meeting details: Division of Infection Journal Club, Barts and The London NHS Trust, London. 5th February 2010. Title: Quantitative-PCR analysis of Fusobacterium nucleatum associated with adverse pregnancy outcomes. Meeting details: The 88th General Session & Exhibition of the IADR. Barcelona, Spain. 14 th – 17 th July 2010. Title: Detection of periodontal bacteria in neonatal gastric aspirates using molecular methods based upon the 16S rRNA gene. Meeting details: Oral Microbiology and Immunology Group meeting. Gregynog, Wales. 10 th – 12 th November 2010. Prize awarded: Postgraduate funding award. Title: Fusobacterium nucleatum associated with adverse pregnancy outcomes. Oral or vaginal origin? Meeting details: Society for Anaerobic Microbiology. The human microbiome in the context of anaerobic infection. London. 3rd – 4th February 2011. Prize awarded: Young Scientist Meeting Grant. 6 Dedication This thesis is dedicated to my family; my dad, mom, and sisters for all their support and encouragement during my career. Special dedication to Patito, Alexito and Camila “Teach them well and let them lead the way” 7 ACKNOWLEDGEMENTS There are a number of people I would like to thank. Without their support this thesis would have not been written. To my dear family: nuclear and extended, who regardless of the distance are always close to my heart; with their acumen and their example they have taught me to be strong and persistent. They always support my decisions making me feel confident, not afraid to take risks and to follow my goals. Special thanks to my supervisors Rob and Dave; they actively supported me throughout my PhD. They have provided me with the best guidance and helped me to develop my best potential. Thanks to both for giving me the opportunity to build up and make my contribution to this area of research. This project would not have been carried out without the important contribution and generous support provided by the UNESCO-L’OREAL partnership. I feel deeply honoured for having being awarded the prestigious UNESCO/L’ORÉAL Co- Sponsored Fellowships for Young Women in Life Sciences 2009. This competitive award is intended to support young women from all around the world to pursue research in a life science project that can contribute to the solution of challenging problems. This is in fact, an important milestone in my scientific career and a huge responsibility as to being an example for women willing to follow the same track. I would like to thank the collaborators for this study, Dr Mike Millar for his kind support; although not completely convinced of our hypothesis, always open to consider our ideas and provide his; and specially, for suggesting and supplying the neonatal samples for the study. Big thanks to Dr Steve Kempley and Miss Anita Sanghi who were on care of the patients that were part of the study; and also thanks to their clinical team for letting me to be part of their activities and facilitate the approach of the women at the NICU. Thanks to Fiona Warburton, Dr Enid Hennessy, and Dr Mark Simmonds for the statistical support. Finally, thanks to all the collaborators for their input in obtaining ethical approval, submitting grants applications, and preparing the manuscripts that arose from this work. 8 As much as important as the clinical aspect of this study was the laboratory aspect; for which I would like to thank a number of people that contributed to it. Special thanks to Dr Anna Tymon and Dr Lena Ciric from the Eastman Dental Institute who taught me the molecular techniques applied in this project. They were always very helpful and supportive and important contributors when it came to troubleshooting. However, due to the distance (and unreliable Hammersmith and City line), Dr Joe Aduse-Opoku and colleagues in the Centre for Infectious Disease and Immunology of the BICMS became good friends and in-house supporters of my work. Thanks as well to Zohir Alosta, MSc student that contributed to obtain some of the results presented in this thesis using the quantitative PCR analysis, and to our lab manager, Steve Cannon who makes sure we can keep our labwork running smoothly. I have to keep expressing my gratitude to other people from external support that not only prodived guidance but became good friends; specially to Dr Yiping Han (Case Western University, Ohio) broadly cited in this project due to her interest in the same field; and to Dr Jaques Izard (The Forsyth Institute, MA) always pointing me in the right direction. To my friends and colleagues; to Lisa and Mandy, for their spiritual guidance, the great encouragement this represented and for sharing their ideas during long converstions with a nice cup of coffee, they taught me many ‘rules of thumb’. To my running buddy Bianca, for her wise advices (in Span-glish),
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