BACTERIAL COMPOSITION OF HUMAN MILK PUMPED AND STORED IN REAL-LIFE CONDITIONS: A RANDOMIZED, CONTROLLED INVESTIGATION A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Sarah Michelle Reyes Paredes December 2019 © 2019 Sarah Michelle Reyes Paredes BACTERIAL COMPOSITION OF HUMAN MILK PUMPED AND STORED IN REAL-LIFE CONDITIONS: A RANDOMIZED, CONTROLLED INVESTIGATION Sarah Michelle Reyes Paredes, Ph. D. Cornell University 2019 Women use a variety of practices to handle and store their pumped breast milk. Little is known about how these practices influence milk’s bacterial composition. We conducted an in-home randomized, controlled, crossover trial and an observational substudy within it. Fifty-two women pumped the same breast twice within 3.5 h, once with their own pumps and collection kits (“own” supplies) and once with a multiuser pump and sterile collection kits (“sterile” supplies). The type of pump supplies used first was randomized. Milk samples were separated into sterile containers and processed within 2 h after expression or after storage in home refrigerators for 2 or 4 d, or in home freezers for up to 30 d with or without refrigeration for the first 4 d. Before women pumped with their own supplies, the following areas were swabbed: areolas, women’s hands, collection kits, pump tubing, and infants’ mouths. Bacterial composition was assessed via aerobic culturing (milk only) and high-throughput sequencing of the V1-V3 region of the 16S rRNA gene from DNA extracted from samples (milk and swabs). Women’s pumping practices were recorded. Culture and 16S analyses revealed the bacterial composition of milk collected with women’s own supplies contained several times more live aerobic and gram-negative bacteria, and >7 times more Proteobacteria than milk collected with sterile supplies. The majority of these extra bacteria likely originated from milk collection kits and survived refrigeration, freezing, or both better than native milk bacteria (bacteria in milk collected with sterile supplies). More rigorous cleaning practices (e.g., sterilization versus washing or rinsing) were associated with significantly fewer bacteria in milk collected with women’s own supplies, including several times lower odds of milk being culture-positive for gram-negative bacteria. This research revealed typical real-life pumping practices changed the bacterial composition of breast milk, most notably by adding live gram-negative bacteria and Proteobacteria. The majority of these extra bacteria likely originated from milk collection kits and survived storage better than native milk bacteria. These findings can contribute to the development of evidence-based recommendations for handling and storing pumped milk pumped. More research is needed to understand the implications of these findings for infant and maternal health. BIOGRAPHICAL SKETCH Sarah Michelle Reyes Paredes earned her B.A. in Independent Studies: International Studies and Human Nutrition in 2009 and her M.S. in Animal Science in 2012, both from the University of Idaho. During her master’s studies, Sarah worked alongside peers who conducted, then published the first study to characterize the human milk microbiome using high-throughput 16S rRNA sequencing. This experience sparked her interest in the relationships between diet, microbes, and health. At the same time, she became a mother and began her breastfeeding journey. Surprised to learn breast milk is often pumped and fed from a bottle, Sarah became interested in how modern breastfeeding practices influenced the human milk microbiome, and whether changes to it influenced maternal or infant health outcomes. In 2013, Sarah was accepted into the doctoral program at Cornell University, where she sought mentorship from Kathleen Maher Rasmussen, ScD. Together with collaborations with her previous lab and new ones at Cornell, Sarah and Dr. Rasmussen designed, secured funding for, and conducted the randomized trial and observational substudy described in this dissertation. Sarah plans to continue a career in maternal and child nutrition with a particular focus on breastfeeding. Her overall goals are twofold: (a) to contribute evidence on the effects of modern breastfeeding practices on maternal and infant health, and (b) to help women achieve their breastfeeding goals by identifying and addressing barriers, especially through commercialization of products intended to preserve the quality of pumped breast milk. iii ACKNOWLEDGMENTS My doctoral journey was far from what I imagined it would be. It began when I was 4 months pregnant with my second child; it is ending now that my third is 14 months old. This journey has felt like a series of comedic tragedies, chock-full of life lessons and far too little sleep. To all my mentors, colleagues, teachers, classmates, and officemates, I sincerely thank you for your role in helping me complete this research. Thank you for pushing me, for encouraging me, and for supporting me through the ups and the downs. I wish you all the best in your personal and professional endeavors. I am particularly indebted to my advisor, Dr. Kathleen M. Rasmussen, for giving me the opportunity to train under her guidance. Kathy is both an incredible scientist and a balanced individual, making time for her personal and professional goals. I am deeply grateful for her guidance, which helped me bring my own research idea to fruition, and for her endless patience in reading draft after draft after draft of my research papers. I am a better individual, scientist, and science communicator because of her mentorship. Next, I would like to acknowledge my dissertation committee members. In particular, I would like to acknowledge Dr. Anthony Hay and Dr. Michelle (Shelley) McGuire, whose support was critical to the success of the interdisciplinary research I conducted. Anthony graciously allowed me to use his wet lab space and provided mentorship on microbiology and leadership. His commitment to my career development is humbling, and I may never fully appreciate all of the ways his mentorship and advocacy shaped me as a scientist and colleague. I am truly grateful for the many lessons he has imparted on me, including but not limited to pronouncing the word stylus. I consider Anthony a wonderful colleague and a true friend. Similarly, Shelley graciously allowed me to train in her lab group at the University of Idaho, iv where I learned the ins and outs of processing human milk samples for high-throughput 16S rRNA sequencing. Working with Shelley’s group felt a bit like a homecoming as I processed samples on the same work benches where I had conducted my master’s research. I would also like to thank the rest of my committee members. Dr. Julia Finkelstein provided irreplaceable guidance on clinical trials documentation and helped guide my understanding of epidemiology. Dr. Patsy Brannon helped broaden my understanding of nutrition and translational science. There are a few people in the three research groups who deserve thanks. First, my deepest thanks to my undergrad, Dainelle Allen, who helped me process and count more than 3,000 culture plates in 84 days! Thanks to Dr. Janet Williams of the McGuire lab, who somehow knows everything, can fix anything, and is pure joy to be around. I learned so much about the human milk microbiome from her. For this I am truly grateful. Thank you to Morgan Potton and Romana Hyde of the McGuire lab, undergrads who helped process nearly 775 milk and swab samples for 16S analyses in just 3 weeks! Finally, I thank my officemates from the Rasmussen research group, Christine Garner, Liz O’Sullivan, Julia Felice, and Jennifer Peregoy, for their endless support and constructive feedback. I am also grateful for my funding sources, the NIH Nutrition Translation Training Grant, a USDA Hatch Grant, the Trainee Travel Fund awarded by the International Society for Human Milk and Lactation and sponsored by the Larsson-Rosenquist Family Foundation, which paid for my stipend, research, and travel to the University of Idaho, where I processed samples for 16S rRNA sequencing. Additionally, I thank Kathleen Rasmussen for providing funds that paid for 16S analyses for the swab samples presented in Chapter 3. Finally, I’d like to thank Medela for v their in-kind contributions to my study, namely loaning me 2 Symphony® Breast Pumps and donating 60 sterile collection kits. I cannot close without mentioning my deep gratitude for my family. I am grateful for my husband, Paúl Reyes, who stood by me for these 6 long years, sacrificing his own goals so I could pursue mine. I would also like to thank my children, Isolde, Naiara, and Joaquin. Each taught me something new about myself, and my role as a mother. So much of my inspiration for breastfeeding research comes from my experiences breastfeeding these kiddos. I’d also like to thank my in-laws, Eugenio Reyes and Alicia Paredes, for making the long journey from Ecuador multiple times to watch the kids and help around the house so I could focus on work. For this I am truly grateful. Last but not certainly not least, I’d like to thank my parents, Kelly and Holly Hulse. As a child, I watched my parents transform their lives for the benefit of their family. They attended university, worked full-time, and raised us 4 children. My father is a first-generation university graduate. My mother, a woman of mixed race who grew up during the Civil Rights era. They imparted on me the most important lessons I’ve learned: the importance of being unfettered by society’s view of where an individual belongs; and to never ever give up. Without these lessons, I would have never had the courage to pursue this degree, let alone finished it. I am proud to say I’m a first-generation doctorate.