Unearthing the Antibacterial Activity of a Natural Clay Deposit by Keith D. Morrison A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved June 2015 by the Graduate Supervisory Committee: Lynda Williams, Co-Chair Stanley Williams, Co-Chair Rajeev Misra Everett Shock Ariel Anbar ARIZONA STATE UNIVERSITY December 2015 ABSTRACT The discovery and development of novel antibacterial agents is essential to address the rising health concern over antibiotic resistant bacteria. This research investigated the antibacterial activity of a natural clay deposit near Crater Lake, Oregon, that is effective at killing antibiotic resistant human pathogens. The primary rock types in the deposit are andesitic pyroclastic materials, which have been hydrothermally altered into argillic clay zones. High-sulfidation (acidic) alteration produced clay zones with elevated pyrite (18%), illite-smectite (I-S) (70% illite), elemental sulfur, kaolinite and carbonates. Low-sulfidation alteration at neutral pH generated clay zones with lower pyrite concentrations pyrite (4-6%), the mixed-layered I-S clay rectorite (R1, I-S) and quartz. Antibacterial susceptibility testing reveals that hydrated clays containing pyrite and I-S are effective at killing (100%) of the model pathogens tested (E. coli and S. epidermidis) when pH (< 4.2) and Eh (> 450 mV) promote pyrite oxidation and mineral dissolution, releasing > 1 mM concentrations of Fe2+, Fe3+ and Al3+. However, certain oxidized clay zones containing no pyrite still inhibited bacterial growth. These clays buffered solutions to low pH (< 4.7) and oxidizing Eh (> 400 mV) conditions, releasing lower amounts (< 1 mM) of Fe and Al. The presence of carbonate in the clays eliminated antibacterial activity due to increases in pH, which lower pyrite oxidation and mineral dissolution rates. The antibacterial mechanism of these natural clays was explored using metal toxicity and genetic assays, along with advanced bioimaging techniques. Antibacterial clays provide a continuous reservoir of Fe2+, Fe3+ and Al3+ that synergistically attack i pathogens while generating hydrogen peroxide (H O ). Results show that dissolved Fe2+ 2 2 and Al3+ are adsorbed to bacterial envelopes, causing protein misfolding and oxidation in the outer membrane. Only Fe2+ is taken up by the cells, generating oxidative stress that damages DNA and proteins. Excess Fe2+ oxidizes inside the cell and precipitates Fe3+- oxides, marking the sites of hydroxyl radical (•OH) generation. Recognition of this novel geochemical antibacterial process should inform designs of new mineral based antibacterial agents and could provide a new economic industry for such clays. ii ACKNOWLEDGMENTS The work that I have presented in my dissertation all stems from Dr. Lynda Williams pioneering work on antibacterial clays that truly embraces interdisciplinary research. I met with Lynda at a Clay Minerals Society conference and instantly knew I wanted to be a part of her research. Throughout my Ph.D. experience Lynda has focused my uncultivated research interests, while giving me the freedom and confidence to pursue my own research endeavors. Lynda always took time out of her schedule to help work through problems in my research and writing, helping me truly understand the ramifications scientific research can have. I will be forever grateful for the skills I have gained working with Lynda, and like her, I hope to pursue ground breaking interdisciplinary research throughout my career. Field work represented a significant portion of my research, where I was challenged with the task of characterizing antibacterial mineral zones in a complex hydrothermal setting. Dr. Stan Williams worked with me in the field, patiently helping me characterize the alteration settings that formed the antibacterial clay zones. Stan’s advice and expertise in the field were fundamental to my success. I would also like to thank Doug Hamilton, Laurel Hamilton and Ray Huckaba for supporting my field work and allowing me to access the deposit. A pivotal transition in my Ph.D. studies occurred when I began working in Dr. Rajeev Misra’s microbiology lab. Rajeev has greatly improved my work ethic and provides a constant source of enthusiasm for research. He freely opened his lab to facilitate much of my work and truly embraced the interdisciplinary nature of this research. Rajeev takes time to help his students on a daily basis, giving them invaluable iii research training and skills. My experience was no different, with Rajeev guiding me through many complex analytical challenges. The skills I have learned in Rajeev’s lab have been invaluable to my Ph.D. and I truly thank him for this. The other faculty members on my Ph.D. committee (Everett Shock and Ariel Anbar) have provided valuable input that has helped me communicate my research to a broad audience. I would like to thank Everett Shock for taking the time to walk me through many thermodynamic calculations that have provided context to my findings. I appreciate Ariel Anbar’s help in communicating my research ideas, which will help me concisely explain ideas to a broad audience. Others who enriched my experiences during my Ph.D. include; Steven Romaniello, Greg Brennecka, Gwyneth Gordon, Amisha Poret-Peterson, Rick Hervig, David Lowry, Robert Roberson, Denis Eberl, Jennifer Underwood, David Metge, Carolina Londono, Ryan and Krista Hutchison, Danny Foley, Sarah Robinson, Cameron Mercer, Andy Ryan and Caren Burgermeister. My experiences with each of these people have been invaluable to my success as a graduate student. I am very grateful for the instruction I received from Tolek Tyliszczak at the Advanced Light Source Synchrotron, Berkeley, CA and STXM sample preparation advice/spectra from Adam Hitchcock. I would especially like to thank Gwyneth Gordon for participating in my Ph.D. defense and providing me with excellent ICP-MS data. Additionally, I would like to thank The Clay Minerals Society, The Geological Society of America and Achievement Rewards for College Scientists for their recognition and funding of my research. My family has been a constant source of support through my academic endeavors. My parents Dave and Colleen Morrison never doubted my abilities throughout this iv journey and I will be forever grateful for this. They have watched me transition through many phases in my life and I am glad that I continue to make them proud. My trips home to visit my sister Madeline’s family have always brought me joy and eased my stress. My brother in law Ernie and his family have always welcomed me into their homes with open arms and I am grateful they are part of my family. I have always looked up to my sister Madeline and her strength and determination have definitely rubbed off on me. My niece’s and nephew (Gwen, Emiko and Ronin) have allowed me to be a kid at heart and place many of life’s challenges into perspective during my Ph.D. I am very thankful to have such a great family and friends who have helped me in this achievement. v TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................................... xi LIST OF FIGURES ............................................................................................................... xii CHAPTER 1 THE DISCOVERY OF ANTIBACTERIAL CLAYS ........................................ 1 1.1 Introduction to the Study of Antibacterial Minerals ................................. 1 1.2 French Green Clays and Mycobacterium .................................................. 4 1.3 Oregon Hydrothermal Clay Deposit ......................................................... 5 1.4 Research Objectives ................................................................................... 6 2 FIELD AREA ....................................................................................................... 8 2.1 Geologic Setting ......................................................................................... 8 2.2 Mineral Sampling..................................................................................... 10 3 THE MINERALOGY OF AN ANTIBACTERIAL CLAY DEPOSIT ........... 19 3.1 Introduction .............................................................................................. 19 3.2 Characterizing Hydrothermal Alteration Systems .................................. 20 3.2.1 High and Low Sulfidation Alteration ...................................... 21 3.2.2 Hydrothermal Mineral Zones ................................................... 22 3.2.3 Clay Mineralogy ....................................................................... 26 3.3 Results ...................................................................................................... 28 3.3.1 Parent Rock............................................................................... 28 3.3.2 Clay Mineralogy ....................................................................... 30 3.3.3 Open Pit Mine Mineralogy ...................................................... 32 vi CHAPTER Page 3.3.4 Sulfur Mine Mineralogy ........................................................... 37 3.3.5 Foster Creek Mineralogy.......................................................... 39 3.3.6 Road Cut Mineralogy ............................................................... 43 3.3.7 Oxygen Isotope Fractionation in Quartz.................................. 45 3.4 Discussion ................................................................................................ 48 3.4.1 Hydrothermal Formation of the OMT Deposit ....................... 48 3.4.2 Mineralogical Variables and Antibacterial Activity ............... 55 3.5 Conclusions .............................................................................................. 56 4 GEOCHEMISTRY AND ANTIBACTERIAL ACTIVITY............................. 59 4.1 Introduction .............................................................................................. 59 4.2 Results ...................................................................................................... 60 4.2.1 Antibacterial Activity and Leachate Chemistry ...................... 60 4.2.2 Metal Solubility and Speciation ............................................... 66 4.2.3 Antibacterial Activity in the Open Pit Mine ............................ 68 4.2.4 Pyrite Size and Redox .............................................................. 69 4.2.5 Cation Exchange Capacity ....................................................... 71 4.2.6 Transmission Electron Microscopy ......................................... 72 4.3 Discussion ................................................................................................ 73 4.3.1 Antibacterial Mineral Zones .................................................... 73 4.3.2 Environmental Microbes .......................................................... 75 4.3.3 Iron Sulfides and Reactive Oxygen Species ............................ 76 4.3.4 Cation Exchange ....................................................................... 77 vii CHAPTER Page 4.3.5 Mineral and Metal Toxicity ..................................................... 79 4.3.6 TEM .......................................................................................... 80 4.4 Conclusions .............................................................................................. 81 5 THE ANTIBACTERIAL MECHANISM ......................................................... 83 5.1 Introduction .............................................................................................. 83 5.2 Results and Discussion ............................................................................ 87 5.2.1 Metal Solubility and Reactive Oxygen Species ...................... 87 5.2.2 Metal Hydrolysis ...................................................................... 90 5.2.3 Bioimaging ............................................................................... 92 5.2.4 Protein Oxidation ..................................................................... 97 5.2.5 Genetic Stress Responses ......................................................... 97 5.3 Conclusions .............................................................................................. 99 6 CONCLUSIONS, APPLICATIONS AND FUTURE RESEARCH ............. 102 6.1 Summary of Findings ............................................................................ 102 6.2 Topical Wound Healing ......................................................................... 103 6.3 Discovery of New Antibacterial Deposits ............................................. 108 7 METHODS ....................................................................................................... 112 7.1 Mineralogy and Solution Chemistry ..................................................... 112 7.1.1 X-ray Diffraction .................................................................... 112 7.1.2 X-ray Fluorescence ................................................................ 113 7.1.3 Secondary Ion Mass Spectrometry ........................................ 114 7.1.4 Cation Exchange Capacity ..................................................... 116 viii CHAPTER Page 7.1.5 Leachate Elemental Analysis ................................................. 117 7.1.6 Ferric and Ferrous Iron Assay ................................................ 117 7.1.7 Hydrogen Peroxide Assay ...................................................... 118 7.1.8 pH, Eh and Mineral Titrations ............................................... 119 7.1.9 Speciation Modeling .............................................................. 119 7.2 Microbiology .......................................................................................... 120 7.2.1 Agar Diffusion Antibacterial Testing .................................... 120 7.2.2 Antibacterial Succeptibility Spot Plating ............................... 121 7.2.3 E. coli Metal Toxicity ............................................................. 122 7.2.4 Metal Solubility ...................................................................... 123 7.2.5 Protein Carbonylation ............................................................ 124 7.2.6 Genetic Stress Responses ....................................................... 125 7.3 Bioimaging ............................................................................................. 126 7.3.1 Transmission Electron Microscopy ....................................... 126 7.3.2 Scanning Transmission Electron Microscopy ....................... 127 7.3.3 Scanning Transmission X-ray Microscopy ........................... 128 7.3.4 Nano Secondary Ion Mass Spectrometry .............................. 129 REFERENCES ..................................................................................................................... 130 APPENDIX A QUANTITATIVE XRD FITS ........................................................................ 142 B QUARTZ TEMPERATURE CALCULATIONS .......................................... 145 C ANTIBIOTIC RESISTANT STRAINS ......................................................... 147 ix