Western University Scholarship@Western Electronic Thesis and Dissertation Repository 4-5-2016 12:00 AM Characterization of microbial communities in the mammary glands and the influence of this microbiota on health and disease Camilla Urbaniak The University of Western Ontario Supervisor Dr. Gregor Reid The University of Western Ontario Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Camilla Urbaniak 2016 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Medical Sciences Commons Recommended Citation Urbaniak, Camilla, "Characterization of microbial communities in the mammary glands and the influence of this microbiota on health and disease" (2016). Electronic Thesis and Dissertation Repository. 3738. https://ir.lib.uwo.ca/etd/3738 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The collection of microbes and their genomic activity within us, referred to as the human microbiome, is crucial in maintaining health, and disruptions in composition and/or activity have been associated with various diseases. Considering the importance of breast milk in neonatal development and the high rate of breast cancer in women, the objective of my thesis was to obtain a comprehensive analysis of the microbiota of the mammary glands and to examine the influence of these organisms on health and disease. Human milk is an important source of bacteria for the developing infant. While a variety of studies over the last 20 years have provided a greater appreciation for how diverse and variable the milk microbiota is, little is known about these bacterial communities. Many studies have emphasized the adverse effects of antibiotics on the infant microbiota, however the impact of drugs on the lactating mother has not been studied. In Chapter 2 we report the changes caused by chemotherapy (over a 4 month period) on the milk microbiota and metabolome of a woman undergoing treatment for Hodgkin’s lymphoma. In Chapter 3, we show from a study of 39 Canadian women that neither gestation, mode of delivery nor gender of the child explains the diversity of the milk microbiota. As milk is not sterile and given the nutrient-rich fatty composition of the breast and its exposure to the external environment via the nipple, we hypothesized that breast tissue has its own indigenous microbiota. In Chapter 4, we confirmed this in a study of breast tissue from 81 Canadian and Irish women, with the discovery of diverse bacterial communities dominated by the phylum Proteobacteria. To determine whether this tissue microbiota could play a role in breast cancer development, we compared, in Chapter 5, ii bacterial profiles in tissue between women with breast cancer and those who were disease free. We show that differences do exist and that women with cancer have higher numbers of bacteria with the ability to induce host DNA damage. This work forms the platform for further studies that examine how breast milk and tissue are first colonized and how these microbiotas can be manipulated to promote optimal maternal and infant health. Keywords Human microbiome, breast milk microbiome, breast tissue microbiome, 16S rRNA gene sequencing, breast cancer, DNA damage iii Co-Authorship Statement Chapter 2: Effect of chemotherapy on the microbiota and metabolome of human milk, a case report I co-designed the study, recruited and collected milk samples from lactating mothers, extracted and amplified the milk DNA for sequencing on the Ion Torrent platform, analyzed 16S rRNA gene sequencing data and wrote the manuscript. Amy McMillan ran the milk samples on GC-MS and performed the metabolome analysis. Chapter 3: Human milk microbiota profiles in relation to birthing method, gestation and infant gender I co-designed the study, recruited and collected milk samples from lactating mothers, extracted and amplified the milk DNA for sequencing on the Illumina MiSeq platform, analyzed 16S rRNA gene sequencing data and wrote the manuscript. Chapter 4: Microbiota of human breast tissue I recruited the Canadian subjects, extracted DNA from the tissue samples and plated tissue homogenates for culture analysis. Joanne Cummins recruited and collected breast tissue from Irish women and extracted DNA from these Irish samples. I amplified the extracted DNA from both the Canadian and Irish samples and pooled the samples in equimolar amounts in preparation for sequencing. I also analyzed the 16S rRNA gene sequencing data and wrote the manuscript. Chapter 5: The microbiota of breast tissue and its association with breast cancer I co-designed the study, recruited subjects, extracted the DNA from the samples, amplified the DNA for sequencing on the Illumina MiSeq platform, analyzed 16S rRNA gene sequencing data, performed the γH2AX DNA damage assay and wrote the manuscript. iv Acknowledgments It is fascinating how small, seemingly unassuming events, can have a substantial impact on an individual’s life. For me, those are memories of late evenings in high school, sitting in the Family room with my Dad while everyone else was asleep, working on math and physics problems together. It is during these late night sessions that I began to appreciate the inquiry process of science and the excitement that comes with questioning the unknown. I would never have been able to do this without you Dad. Your versatile talents never cease to amaze me- you are a computer engineer with a PhD in Physics, who can remodel a full kitchen and basement like an expert and whom can fix any problem or answer any question that I have. I hope that one day I can be at least half as talented as you. To mom: Thank-you for dedicating your time and energy into allowing me to experience so many facets of life. Because of you I am creative, compassionate and adventurous. No matter what roadblocks come my way, I am well equipped to deal with them because of you. To Jahan: I cannot express how lucky I am to have met you in undergrad. Your wit and humour has had me doubled over in laughter more times then I can count. I could not imagine my life without you. Thank-you for everything! To Jane: Thank-you for being my rock and my shoulder to complain on. Thank-you for being there through all the tough choices and decisions I have had to make. Your zest for life is infectious and I hope that we continue to have many more adventures together. v To Ania: You are one of the smartest people I know (along with Jane). You have been a constant source of knowledge and feedback throughout the years. Thank-you for being receptive of my “thought” questions and a friend I can trust with all the moments in my life, good or bad. I hope we continue to be friends for many more years to come. To Jorum: Who would have thought that when I walked through the front door of the lab in 2005 we would become such great friends. You have been my best supporter, champion and advocate and have believed in me during times when I didn’t believe in myself. There are not enough words to express my gratitude for all your kindness. I hope that one day we can again work together. There have been so many people that have come into my life throughout the years. Whether it has just been in passing or for a longer period of time, you have all made a difference in my life and have made me who I am today. Finally, I would like to thank my committee members, Muriel Brackstone, Greg Gloor and Jim Koropatnick for their mentorship and guidance throughout my PhD. I would especially like to thank my supervisor, Gregor Reid, for his support. His dedication to his students is truly inspirational and without him I would not have learned and have achieved as much as I have. You are one of a kind and I am truly blessed to have been able to do a PhD with you. ~“Many of life’s failures are people who did not realize how close they were to success when they gave up” - Thomas A. Edison~ vi Table of Contents Abstract ............................................................................................................................... ii Co-Authorship Statement ................................................................................................... iv Acknowledgments ............................................................................................................... v Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii List of Abbreviations ....................................................................................................... xvi Chapter 1 ............................................................................................................................. 1 1 General introduction ....................................................................................................... 1 1.1 Functional anatomy of the female breast ................................................................ 1 1.1.1 Embryogenesis ...........................................................................................