UNIVERSITY OF CALIFORNIA, IRVINE The Effects of Recreational Water Exposure on Human Skin: Toxin Penetration and Microbiome Alteration DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Environmental Health Sciences by Marisa Chattman Nielsen Dissertation Committee: Professor Sunny Jiang, Chair Professor Scott Bartell, Professor Ulrike Luderer Professor Oladele Ogunseitan 2020 © Marisa Chattman Nielsen DEDICATION To my parents, Joann and Martin Chattman, for their unconditional love and support. To my husband, Barton Nielsen, I could not have done this without you. Thank you for your encouragement. To my son, Bradford Nielsen, thank you for helping me realize that if I can write a dissertation with a 2 year old on my lap, I can do anything. To my brother, Jacob Chattman, for your guidance. Unfortunately, there was no opportunity for your creative input in this dissertation. To Michael Johnson, thank you for believing in me and reminding me every Thanksgiving for the past 10 years that I needed to do this. ii TABLE OF CONTENTS Page LIST OF FIGURES v LIST OF TABLES vi ACKNOWLEDGMENTS vii CURRICULUM VITAE viii ABSTRACT OF THE DISSERTATION ix INTRODUCTION 1 CHAPTER 1: Understanding the Risk of Cyanotoxin Skin Penetration during Recreational Water Exposure 6 Graphical Abstract 7 Introduction 7 Cyanobacteria blooms in recreational water 8 Common cyanotoxins 10 Current recreational water exposure guidelines 14 Dermal exposure toxicology studies 17 Epidemiological studies 20 Permeation through the skin 25 Skin characteristics affecting permeation 28 Summary and future directions 30 CHAPTER 2: Alterations of the Human Skin Microbiome after Ocean Water Exposure 34 Abstract 35 Introduction 35 Methods 40 Results 43 Discussion 51 CHAPTER 3: Changes in the antibiotic resistant gene profile of the skin microbiome in response to ocean water exposure 57 Introduction 58 Methods 63 Results 67 Discussion 80 CONCLUSIONS 84 iii APPENDIX A: Supporting material for Chapter 2 86 REFERENCES 89 iv LIST OF FIGURES Page Figure 1.1 Mean levels of cyanotoxins in US lakes by month 10 Figure 1.2 Lowest Recreational Water Action Level for specific cyanotoxins by individual State in the U.S. based on EPA guidelines 17 Figure 1.3 Plot of five cyanotoxins (MC-LR, nodularin, anatoxin-a, saxitoxin and cylindrospermopsin) according to their logP values and molecular mass for prediction of skin permeation. 27 Figure 2.1 The bacterial community alpha diversity of skin microbiome samples 44 before and after ocean swimming Figure 2.2 The bacterial community beta diversity of the skin microbiome before and after ocean swimming 46 Figure 2.3 Microbial community composition of the skin microbiome 48 Figure 2.4 Changes in microbial composition before and after ocean exposure 50 Figure 3.1 Network analysis showing the relationship among bacterial phyla and ocean water exposure in human skin microbiome samples 69 Figure 3.2 Sub-types of predicted ARGs before and after ocean water exposure 70 Figure 3.3 Heatmap of ARGs, categorized by antibiotic sub-type, present on human skin before and after ocean water exposure 72-73 Figure 3.4 Predicted antibiotic biosynthesis gene count 75 Figure 3.5 Heatmap of VFG KO’s present on human skin before and after ocean water exposure 77 Figure 3.6 Occurrence, abundance and diversity of ARGs 79 v LIST OF TABLES Page Table 1.1 EPA and WHO algal toxin guidelines 16 Table 1.2 Summary of human health effects described in case studies of exposure to contaminated recreational water 24 Table 3.1 Antibiotics and their corresponding ARGs and KOs 66 Table 3.2 Median values for ARG count in each sub-type 71 Table 3.3 Median values for ABSG count in each sub-type 75 vi ACKNOWLEDGMENTS It is an honor to express my deepest appreciation to my committee chair, Professor Sunny Jiang, who is not only an incredible mentor and advisor, but she is a wonderful friend. She has been so supportive and understanding throughout my time in her lab. She truly cares about her students and I am proud to represent her lab as I continue forward in my career. I would like to thank my committee members, Professor Scott Bartell, Professor Ulrike Luderer and Professor Oladele Ogunseitan. Dr. Bartell taught me the basics of biostatistics and gave me my first glimpse into the computer program, R, which I have used for countless hours since. Dr. Luderer helped shape my public speaking and presentation skills, so that I am now a confident public speaker, unafraid to present my research to a room full of scholars. Dr. Ogunseitan gave me such a warm welcome when I first visited UCI during interview day and his support continued throughout my time at UCI. I am grateful for all the guidance and mentorship I have received from the entire Environmental Health Sciences program. Financial support was provided by the University of California, Irvine, NSF CBET 1806066, 1804166 and UCI Oceans Fellowship. vii CURRICULUM VITAE Marisa Chattman Nielsen EDUCATION 2020 Ph.D. in Environmental Health Sciences. University of California. Irvine, CA. 2006 M.S. in Soil, Water and Environmental Science. University of Arizona. Tucson, AZ. 2004 B.S. in Microbiology. University of Arizona. Tucson, AZ PROFESSIONAL EXPERIENCE 2015-2020 Clinical Laboratory Scientist in Microbiology. Hoag Hospital. Newport Beach, CA. 2013-2015 Supervisor of the Clinical Microbiology Laboratory. Tufts Medical Center. Boston, MA. 2012-2013 Assistant Supervisor of the Clinical Microbiology Laboratory. Boston Medical Center. Boston, MA. 2008-2012 Medical Technologist II in the Clinical Microbiology Laboratory. Boston Medical Center. Boston, MA PUBLICATIONS Nielsen Chattman M. and Jiang S. Alterations of the human skin microbiome after ocean water exposure. Mar Pollut Bull 2019, 145:595–603. Chattman M., Maxwell S. L., Gerba C. P. Occurrence of heterotrophic and coliform bacteria in liquid hand soaps from bulk refillable dispensers in public facilities. J. Environ. Health 2011, 73:26–29. viii ABSTRACT OF THE DISSERTATION The Effects of Recreational Water Exposure on Human Skin: Toxin Penetration and Microbiome Alteration By Marisa Chattman Nielsen Doctor of Philosophy in Environmental Health Sciences University of California, Irvine, 2020 Professor Sunny Jiang, Chair Skin is the body’s first line of defense against the external environment and exposure to recreational water can compromise the skin’s protective functions. Recreational water often contains harmful algal blooms, cyanotoxins, pathogenic bacteria, antibiotics and antibiotic resistance genes. This research investigated the following effects of recreational water exposure on human skin: cyanotoxin skin penetration potential, changes in the human skin microbiome and acquisition of exogenous antibiotic resistance genes (ARGs), antibiotic biosynthesis genes (ABSGs) and virulence factor genes (VFGs). Cyanotoxin penetration potential was investigated in an in-depth examination of the state of knowledge on cyanotoxins and their potential to cause negative health effects through dermal permeation. Epidemiological and toxicological studies of the health effects from algal toxin exposure are summarized to highlight the importance of better understanding of the effects on human skin. This research identified a disparity between the human health effects described in ix epidemiology case studies and toxicological dermal exposure data, indicating potential dermal penetration. The penetrative abilities of specific cyanotoxins were predicted by their physiochemical properties indicating the potential for skin penetration. These predictions can be used to better evaluate human health risks. Another component of the skin’s protective role is the microbiome, which has been shown to provide immunity against exogenous bacterial colonization. This study explored the link between ocean water exposure and the human skin microbiome, and demonstrated that there are post-exposure alterations. Skin microbiome samples were collected from human participants’ calves before and after they swam in the ocean, and at 6 hours and 24 hours post-swim, and were analyzed using 16s rRNA gene and metagenomic sequencing. Beta diversity analysis revealed that the skin microbial communities on all participants before swimming were different from one another, but immediately after swimming, all participants’ microbial communities were tightly clustered, indicating that the communities were no longer different. Taxonomic analysis showed that ocean bacteria, including potential pathogens, replaced the native skin bacteria and remained on the skin for at least 24 hours post-swim. Metagenomic analysis and functional gene predictions showed that ARGs, ABSGs and VFGs present on the skin increased in diversity and abundance after participants swam in the ocean and persisted for at least 6 hours post-swim. This research provides insight into the relationship between human health, the skin microbiome and the environment. x INTRODUCTION The skin is a complex organ responsible for protecting the body from physical, chemical and biological insults. Its extensive structure is organized into the epidermis (top layer) and the dermis (bottom layer), which are separated by the basement membrane [1]. Hair follicles, sebaceous glands, sweat ducts and in some body sites apocrine glands, span the epidermal and dermal layers [2]. These layers and structures create a complex environment that not only serves as a protective barrier but also sustains a variety of commensal and pathogenic