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Antimicrobial properties of Kisameet clay, a natural clay mineral from British Columbia, Canada Behroozian, Shekooh 2019

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ANTIMICROBIAL PROPERTIES OF KISAMEET CLAY, A NATURAL CLAY MINERAL FROM BRITISH COLUMBIA, CANADA by Shekooh Behroozian B.Sc., University of Isfahan, 1992M.Sc., Shiraz University of Medical Sciences, 2003A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2019 © Shekooh Behroozian, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Antimicrobial Properties of Kisameet Clay, A Natural Clay Mineral from British Columbia, Canada submitted by Shekooh Behroozian in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Examining Committee: Dr. Julian E. Davies Co-supervisor Dr. Michael E.P. Murphy Co-supervisor  Dr. Charles R. Thompson Supervisory Committee Member Dr. Thomas J. Beatty University Examiner Dr. Katherine S. Ryan University Examiner Additional Supervisory Committee Members: Dr. Pauline Johnson Supervisory Committee Member iii Abstract      The dearth of new antibiotics and the widespread emergence of multi-drug resistant (MDR) bacteria have created a global crisis in medicine and highlighted the drastic need for novel antimicrobial agents. Natural clay minerals with a long history of therapeutic and biomedical applications have recently received increasing attention due to recent studies on their potent antibacterial activity. Kisameet clay (KC), a natural clay deposit found in British Columbia, Canada, has been used by local First Nation people for medicinal purposes for generations. This research investigates the antimicrobial properties of KC, the spectrum of activity, and the mechanism(s) underlying its antibacterial action. In order to characterize KC antimicrobial activity and define its active components, a series of integrated microbiological, chemical, and mineralogical studies have been performed.         This study revealed that aqueous suspensions of KC exhibit in vitro broad-spectrum antimicrobial activity against a variety of MDR bacterial pathogens, including the ESKAPE pathogens and Cystic fibrosis clinical isolates. Moreover, two major fungal pathogens, Candida albicans and Cryptococcus neoformans were also susceptible to KC. In addition, KC aqueous leachates (KC-L) show potent bactericidal activity in which low-pH plays a key role. Treatment of KC minerals and KC-L with cation-chelating agents indicates roles for divalent and trivalent cations, more specifically iron and aluminum. Further studies suggest that the bactericidal activity of KC-L is due to multiple modes of action. The low-pH buffered environment, rich in a combination of released metal ions, can synergistically challenge treated bacteria to maintain their metal homeostasis, while aluminum-related impairment of outer membrane (OM) permeability may exacerbate this situation. KC-L can, concurrently, stress multiple bacterial components, cause metal intoxication and consequential cell damage, impair OM and destabilize the cell membrane structure. Furthermore, it induces oxidative stress, generates hydrogen peroxide, and damages DNA, which collectively leads to lethal pleiotropic effects in treated bacteria. Further studies detected KC-L-related transcriptional modulation in oxidative- and envelope- stress responses, DNA iv damage, metal detoxification pathways, or efflux pump function. Better understanding of the principal components of KC antibacterial activity may permit the formulation of defined, active preparations of this natural clay mineral for therapeutic applications.  v Lay Summary      The discovery and development of antimicrobials as therapeutic agents has transformed modern medicine. However, their further mass production, global distribution, and gross misapplication have resulted in escalating emergence of multi-drug resistant (MDR) bacteria worldwide. Given the limited arsenal of antimicrobials available to combat MDR bacteria, there is growing apprehension about the threat posed by “superbugs” and a pressing need for novel therapeutics. Natural clay minerals have a long history of therapeutic and biomedical applications and lately received increasing attention for their antimicrobial properties. Kisameet clay (KC), a naturally-occurring clay mineral, has been long known for its therapeutic properties. Herein, the potent and broad-spectrum antimicrobial activities of KC against a variety of MDR bacterial and fungal pathogens have been described. The findings presented in this study provide a better understanding of the principal components of KC antibacterial activity and create a basis to develop defined preparations of KC for therapeutic applications.  vi  Preface       This statement confirms that the author of this thesis is the primary person responsible for the design and initiation of this research project with direct input from supervisors, Dr. Julian Davies, and Dr. Michael Murphy. My thesis committee members, Dr. Charles Thompson and Dr. Pauline Johnson as well as Dr. Vivian Miao also made considerable contributions to the design of this research.        Chapter 2 on the characterization of KC is based on work conducted in UBC’s Department of Microbiology and Immunology, the Davies laboratory and the Department of Civil Engineering. Mineralogical studies, ICP-OES analysis of acid-digested KC core samples and redox potential measurements were carried out by Dr. Wanjing Xu in Dr. Loretta Li’s laboratory in Civil Engineering, while investigation of all other physicochemical properties of KC core samples as well as their antibacterial activity, presented in this chapter, were carried out by me and Dr. Sarah Svensson in the Davies Laboratory. In addition, undergraduate students Ahmad Maslati, and Kevin Ye received training from me and assisted with isolation of bacterial strains from KC samples. 16S rRNA gene sequencing studies have been carried out by me, Ahmad Maslati, and Miguel Desmarais in the Davies Laboratory. Some content from Chapter 2 has been published in a different format in Svensson et al., 2017.     The antimicrobial assays presented in chapters 3, 4, and 5 have been conducted by me in the Davies laboratory. I designed and performed all experiments for this chapter under Dr. Julian Davies and Dr. Michael Murphy’s supervision with the following exceptions:      ICP-OES analysis, presented in chapter 3 was performed in the Department of Civil Engineering with the assistance of Dr. Wanjing Xu and Dr. Loretta Li, while, the ICP-MS analysis reported in Chapter 4 was performed in the Murphy Laboratory in the Department of Microbiology and Immunology in UBC by Mariko Ikehata. In addition, Transmission electron microscopy (TEM) studies were performed in Bioimaging Center at UBC; however, Dr. John Smit (UBC) helped me considerably to load the bacterial samples on grids for further TEM studies. For oxidative stress studies presented in Chapter 5, Dr. James  vii  Imlay from the Department of Microbiology in the University of Illinois provided me with the E. coli mutant strains and also provided their lab procedure for catalase supplementation of media.      Some content from Chapter 3 has been published in Behroozian et al., 2016, while two manuscript versions of the rest of data presented in this chapter are ready for submission. Behroozian S, Svensson SL, Xu W, Li L, Davies J. Broad-spectrum antimicrobial and anti-biofilm activity of Kisameet clay, a natural clay mineral. In this manuscript all the experiments have been designed and carried out by me, while Dr. Sarah Svensson performed the biofilm studies which are just included in the manuscript, not this thesis. Dr. Wanjing Xu performed the mineralogical analyses. Behroozian S, Zlosnik JEA, Davies J. Antibacterial activity of Kisameet clay against Burkholderia cepacia complex and other bacterial pathogens isolated from patients with Cystic Fibrosis. The experiments presented in this manuscript have been all designed and performed by me under the supervision of Dr. Julian Davies in the Davies Laboratory, while Dr. James Zlosnik from Department of Pediatrics, Centre for Understanding and Preventing Infection in Children, B. C. Children’s Hospital Research Institute, UBC, provided me with the Cystic fibrosis related clinical pathogens. List of publications arising from my thesis work:  Svensson SL, Behroozian S, Xu W, Surette MG, Li L, Davies J. 2017. Kisameet glacial clay: an unexpected source of bacterial diversity. mBio 8:e00590-17.  Behroozian S, Svensson SL, Davies J. 2016. Kisameet clay exhibits potent antibacterial activity against the ESKAPE pathogens. mBio 7(1):e01842-15.     viii  Table of Contents  Abstract ....................................................................................................................................................... iii Lay Summary .............................................................................................................................................. v Preface ......................................................................................................................................................... vi Table of Contents ..................................................................................................................................... viii List of Tables ............................................................................................................................................ xiv List of Figures ............................................................................................................................................ xv List of Abbreviations ............................................................................................................................. xviii List of Symbols ....................................................................................................................................... xxiv Acknowledgements ................................................................................................................................. xxv Dedication .............................................................................................................................................. xxvii  Introduction and Literature Review ...................................................................................... 1  Infections, antibiotics, and antibiotic resistance .......................................................................... 1  Urgent need for novel antimicrobial agents ................................................................................. 2  Alternative antimicrobial agents .................................................................................................. 4  Natural medicinal clay minerals, their existence in human life since prehistory .................... 5  Traditional applications of medicinal clay minerals ....................................................... 5  Modern applications for prophylactic and therapeutic purposes .................................... 6  Clay and clay minerals, nanomaterials of geological origin ........................................... 7  Clay minerals, classification and main characteristics ................................................... 8  Antibacterial clay minerals: a decade of studies........................................................... 13  Kisameet clay, history of therapeutic applications .................................................................... 15  Antibacterial activity reported in 1946 .................................................................................. 19  Research objectives ................................................................................................................... 20  ix   Characterization of Kisameet Clay ...................................................................................... 21  Introduction ............................................................................................................................... 21  Materials and methods ............................................................................................................... 21  Clay sample collection and preparation ................................................................................ 21  Measurement of pH and redox potential ............................................................................... 23  Mineralogical composition by X-ray diffraction ................................................................... 23  Aqueous leachate preparation ............................................................................................... 23  Elemental analysis of bulk clay samples and aqueous leachates by inductively coupled plasma optical emission spectrometry (ICP-OES) ............................................................................. 24  Antibacterial activity of vertical core samples ...................................................................... 24  Isolation of KC resident bacteria using different selective and differential media ............... 25  Identification of KC bacterial isolates by 16S rRNA sequencing ......................................... 25  Results ....................................................................................................................................... 27  KC vertical cores and bucket samples ................................................................................... 27  Mineralogical compositions .................................................................................................. 29  Physicochemical properties of KC samples including pH, redox, and elemental analyses .. 29  Biological properties ............................................................................................................. 37  KC resident bacteria ..................................................................................................... 38 2.3.4.1.1 KC resident bacteria identified by 16S rRNA sequencing ..................................... 38 2.3.4.1.2 Phylogenetic tree of KC bacterial isolates.............................................................. 39  Antibacterial activity of KC samples among vertical cores ......................................... 39  Discussion .................................................................................................................................. 43  Antimicrobial Properties of Kisameet Clay ........................................................................ 45  Introduction ............................................................................................................................... 45  Materials and methods ............................................................................................................... 45  x   Microbial strains and growth conditions ............................................................................... 45  Kisameet clay samples .......................................................................................................... 49  KC suspensions and aqueous leachates preparations ............................................................ 49  Elemental analysis of KC aqueous leachates by ICP-OES ................................................... 49  Antibacterial agents and susceptibility assays....................................................................... 50  Antimicrobial assays of KC aqueous suspensions and leachates .......................................... 52  Viability assays ............................................................................................................. 52  Growth inhibition assays for M. marinum .................................................................... 52  Organic solvent extracts of KC ............................................................................................. 53  Results ....................................................................................................................................... 53  Antibacterial activity of KC aqueous suspensions ................................................................ 53  Antibacterial activity of KC aqueous leachates .................................................................... 54  Antibacterial activity of KC organic extracts ........................................................................ 57  Antibacterial activity of KC against clinical MDR bacterial isolates ................................... 59  ESKAPE pathogens ...................................................................................................... 59 3.3.4.1.1 ESKAPE pathogens and their clinical importance ................................................. 59 3.3.4.1.2 Antibiotic resistance patterns of ESKAPE pathogens ............................................ 60 3.3.4.1.3 Antibacterial activity of KC suspensions against ESKAPE pathogens .................. 60  Burkholderia cepacia complex, P. aeruginosa, and S. maltophilia isolated from cystic fibrosis patients .............................................................................................................................. 64 3.3.4.2.1 Cystic fibrosis ......................................................................................................... 64 3.3.4.2.2 Antibacterial resistance patterns of CF clinical isolates ......................................... 65 3.3.4.2.3 Antibacterial activity of KC suspensions and aqueous leachates against MDR clinical pathogens isolated from CF patients ............................................................................. 68  Antibacterial activity of KC against M. marinum as a model of M. ulcerans ....................... 72  xi   Antifungal activity of KC suspensions against C. albicans and C. neoformans ................... 72  Discussion .................................................................................................................................. 74  Roles of pH and Metal Ions in the Antibacterial Activity of KC ...................................... 79  Introduction ............................................................................................................................... 79  Materials and methods ............................................................................................................... 82  Bacterial strains and growth conditions ................................................................................ 82  Chelation assays .................................................................................................................... 82  Treatment of KC suspensions with chelating agents .................................................... 82  Treatment of KC leachates with chelating agents ........................................................ 83  pH adjustment of aqueous leachates and precipitation assays .............................................. 83  Elemental analysis by ICP mass spectrometry (ICP-MS) ..................................................... 84  Preparation of MES- buffered defined minimal medium (MBMM) ..................................... 84  Preparation of single metal ion solutions (FeCl2, FeCl3, AlCl3) and metal ion mixture (MIM) ………………………………………………………………………………………………85  Antibacterial activity of metal ion solutions on the viability and growth of bacteria ........... 86  Determining minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) of metal ion solutions .............................................................................................................. 86  Results ....................................................................................................................................... 87  Effect of cation chelating agents on the antibacterial activity of KC mineral and leachate .. 87  Role of pH in the antibacterial activity of KC....................................................................... 94  pH adjustment experiments and precipitate formation ................................................. 95  Elemental analyses of precipitates by ICP-MS and ICP-OES ...................................... 98  Antibacterial activity of single metal ion (Fe2+, Fe3+, Al3+) solutions and metal ion mixture (MIM) on the viability and growth of bacteria ................................................................................... 99  MIC and MBC of metal ion solutions ................................................................................. 103  xii   Discussion ................................................................................................................................ 104  Elucidating the Mode(s) of Action of KC Leachates ........................................................ 107  Introduction ............................................................................................................................. 107  Materials and methods ............................................................................................................. 109  Bacterial strains and growth conditions .............................................................................. 109  Antibacterial assays ............................................................................................................. 111  Antibacterial activity of KC leachate under aerobic vs. anoxic condition ................. 111  Catalase supplementation of growth media ................................................................ 111  Antibacterial activity of KC leachate against oxidative stress related bacterial mutants ……………………………………………………………………………………….112  Transmission electron microscopy (TEM) studies .............................................................. 113  Outer membrane permeability assay ................................................................................... 113  BacLight Live/Dead assay and flow cytometry .................................................................. 114  Transcriptional changes induced by KC leachate on an E. coli reporter library ................. 115  Results ..................................................................................................................................... 116  Role of oxidative stress ....................................................................................................... 116  Comparative antibacterial activity of KC leachate under aerobic vs. anoxic condition ……………………………………………………………………………………….116  Effect of catalase supplementation on the sensitivity of bacteria to KC leachate ...... 117  Susceptibility of bacterial strains with mutations in oxidative-stress related functions to KC leachate .................................................................................................................................. 118  Susceptibility of bacterial strains with mutations in DNA repair related functions to KC leachate .................................................................................................................................. 120  Effect of KC leachate on cell membrane integrity and permeability .................................. 121  Ultrastructural studies of treated bacteria by TEM ..................................................... 121  xiii   Effect of KC leachate on the OM permeability .......................................................... 125  Effect of metal ions and artificial metal ion mixture on the OM permeability .......... 126  Effect of KC leachate on the CM integrity of treated cells ........................................ 127  Transcriptional modulations upon treatment with KC leachate .......................................... 129  Discussion ................................................................................................................................ 132  Conclusions and Future Directions .................................................................................... 137  The pressing need for novel antimicrobial agents and innovative strategies........................... 137  The complex nature of KC ...................................................................................................... 137  Broad-spectrum antimicrobial properties of KC ..................................................................... 138  Metal ion toxicities in a pH-dependent manner ....................................................................... 140  OM permeabilization and CM impairment upon KC leachate treatment ................................ 142  Multi-target mechanism of action ............................................................................................ 143  Significance of this study ........................................................................................................ 145 References ................................................................................................................................................ 146 Appendices ............................................................................................................................................... 173 Appendix A .......................................................................................................................................... 173 A.1 Culture Media Composition ................................................................................................ 173 A.2 Bacterial Strains Isolated From KC Samples ...................................................................... 174 Appendix B ........................................................................................................................................... 178 B.1 Metal Stability Constants of Cation Chelators .................................................................... 178 Appendix C ........................................................................................................................................... 179 C.1 Sensitivity of E. coli Mutants with Defect in Iron-Regulation or -Storage Functions to KC Leachate ........................................................................................................................................... 179 Appendix D .......................................................................................................................................... 182 D.1 Description of GFP Clones of E. coli MG1655 Summarized in Fig. 5.11. ......................... 182  xiv  List of Tables  Table 2.1 Mineralogical composition of selected KC core samples ........................................................... 31 Table 2.2 Physicochemical properties of KC samples based on depth ....................................................... 32 Table 2.3 Acid-digested elemental composition of KC bulk clay samples ................................................ 33 Table 2.4 117 KC isolates identified by 16S rRNA gene sequencing ........................................................ 40 Table 3.1 Bacterial and fungal strains used in this study ............................................................................ 47 Table 3.2 Clinical pathogens isolated from cystic fibrosis patients ............................................................ 48 Table 3.3 Antibacterial agents used in this study ........................................................................................ 51 Table 3.4 Elemental analysis of KC aqueous leachates (L50, L100, and L500) by ICP-OES ................... 58 Table 3.5 Antibacterial activities of KC organic extracts against four bacterial strains ............................. 59 Table 3.6 Resistance patterns of ESKAPE strains for different classes of antibiotics ................................ 61 Table 3.7 Resistance patterns of CF isolates to different classes of antimicrobial agents .......................... 67 Table 4.1 pH comparison of KC aqueous suspensions and leachates treated with EDTA, 2,2ˈ-bipyridyl (BPY), or deferoxamine (DFO) .................................................................................................................. 92 Table 4.2 Elemental composition of KC leachates, L50, L100, and L500. Analyte levels were determined by ICP-MS .................................................................................................................................................. 93 Table 4.3 ICP-MS analysis of KC-L (L50, L100), and supernatants after collecting the precipitates ....... 98 Table 4.4 MIC and MBC values of single metal ion solutions for three bacterial strains growing in MBMM broth ............................................................................................................................................ 103 Table 4.5 MIC and MBC values of metal ion solutions for E. coli MG1655 growing in four different broth media ............................................................................................................................................... 103 Table 5.1 Oxidative-stress and DNA-damage related mutant strains of E. coli MG1655 used in this study ................................................................................................................................................................. 110  xv  List of Figures  Figure 1.1 The timeline of antibiotic discovery illustrating the history of discovery and the subsequent discovery void ............................................................................................................................................... 3 Figure 1.2 Classification of silicate minerals .............................................................................................. 10 Figure 1.3 Pharmaceutical and biomedical applications ............................................................................. 11 Figure 1.4 The Kisameet Bay glacial clay deposit ...................................................................................... 18 Figure 2.1 Map of the Kisameet Bay glacial clay deposit, spatial description of vertical core sampling .. 22 Figure 2.2 Examples of Kisameet clay samples ......................................................................................... 28 Figure 2.3 Light microscopy photographs of KC particles, presence of large silica and mica-type particles (X400) .......................................................................................................................................................... 29 Figure 2.4 Chemical properties of KC samples from different depths compared to KC35 ........................ 36 Figure 2.5 Bacterial diversity among KC samples...................................................................................... 38 Figure 2.6 Neighbour-joining tree based on the V2, V3, and V4 regions of 16S rRNA gene sequences, showing the phylogenetic relationship among the KC isolates .................................................................. 42 Figure 2.7 Antibacterial activity of suspensions of KC core samples against E. coli MG1655 ................. 43 Figure 3.1 Viability of E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) after treatment with 1%  (wt/vol) aqueous suspensions of KC ........................................................................... 54 Figure 3.2 Antibacterial activity of three KC aqueous leachates (L50, L100, and L500) against E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) compared to low-pH phosphate buffers ......................................................................................................................................................... 57 Figure 3.3 Effect of 1% (wt/vol) aqueous suspensions of KC on the viability of various ESKAPE strains .................................................................................................................................................................... 63 xvi Figure 3.4 Effect of aqueous suspensions of KC on the viability of CF isolates: 10% (wt/vol) against B. cepacia complex isolates (A-C) and 1% (wt/vol) against P. aeruginosa isolates (D) and S. maltophilia (E) .................................................................................................................................................................... 69 Figure 3.5 Effect of KC-L (L100) on the viability of B. cepacia complex isolates (A-C), P. aeruginosa isolates (D), and S. maltophilia (E) ............................................................................................................. 71 Figure 3.6 Antibacterial activity of KC against M. marinum ..................................................................... 72 Figure 3.7 Effect of 5% (wt/vol) aqueous suspensions of KC on the viability of fungal strains, C. albicans SC5314 (A) and C. neoformans H99 (B) .................................................................................................... 73 Figure 4.1 Viability of E. coli MG1655 in 1% (wt/vol) aqueous suspension of KC pre-washed with 10 or 100 mM EDTA (A) and KC-L (L50) treated with 1 mM EDTA and pH adjustment (B) .......................... 89 Figure 4.2 Viability of E. coli MG1655 in 1% (wt/vol) aqueous suspension of KC pre-washed with 10 mM 2,2ˈ-bipyridyl (BPY) (A) and in KC-L (L50) treated with 1 mM BPY and pH adjustment (B). ........ 90 Figure 4.3 Viability of E. coli MG1655 in 1% (wt/vol) aqueous suspension of KC prewashed with 10 mM deferoxamine (DFO) (A) and in KC-L (L50) treated with 1 mM DFO and pH adjustment....................... 92 Figure 4.4 Effect of pH on the antibacterial activity of KC-L (L50) .......................................................... 94 Figure 4.5 KC-L (L50, L100) with the precipitates formed after pH-adjustment to 7.0 (A), collected precipitates after centrifugation (B) ............................................................................................................ 95 Figure 4.6 Antibacterial activity of supernatant (A), and resuspended precipitate (B) compared to the original KC-L (L50) against E. coli MG1655 ............................................................................................. 96 Figure 4.7 Antibacterial activity of supernatant (A) and resuspended precipitate (B) compared to the original KC-L (L100) against E. coli MG1655 ........................................................................................... 97 Figure 4.8 Effect of KC-L (L100) compared to single metal ion solutions (FeCl2, FeCl3, and AlCl3) and metal ion mixture (MIM) on the viability of E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) ............................................................................................................................... 100  xvii  Figure 4.9 Antibacterial activity of KC leachate (L100) compared to single metal ion solutions (FeCl2, FeCl3, and AlCl3) and metal ion mixture (MIM) on the growth E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) in a defined-minimal medium (MBMM) ........................................... 102 Figure 5.1 Comparative antibacterial activity of KC-L (L100) against E. coli MG1655 under aerobic vs. anoxic conditions ...................................................................................................................................... 116 Figure 5.2 Effect of catalase-supplementation on the antibacterial activity of KC-L (L100) against E. coli MG1655 (WT) .......................................................................................................................................... 117 Figure 5.3 Sensitivity of E. coli mutants with defects in oxidative-stress related functions to KC-L (L100) .................................................................................................................................................................. 120 Figure 5.4 Sensitivity of E. coli mutants with defects in DNA repair to KC-L (L100) ............................ 121 Figure 5.5 Time series TEM photographs of E. coli MG1655 cells treated with KC-L (L500) compared to controls ...................................................................................................................................................... 122 Figure 5.6 Time series TEM photographs of S. aureus RN4220 cells treated with KC-L (L500) compared to controls ................................................................................................................................................. 123 Figure 5.7 TEM images of morphological changes induced in E. coli MG1655 (A, B), and S. aureus RN4220 (C, D) as the results of KC-L (L500) treatments ........................................................................ 124 Figure 5.8 Effect of KC-L (L100) on bacterial OM permeability measured by NPN uptake assay ......... 125 Figure 5.9 Effect of metal ion solutions (AlCl3, FeCl2, FeCl3) and metal ion mixture (MIM) compared to that of KC-L (L100) on bacterial OM permeability measured by NPN uptake assay .............................. 126 Figure 5.10 Flow cytometric analysis of E. coli MG1655 treated with dH2O, KC-L (L100), and low-pH buffer ......................................................................................................................................................... 128 Figure 5.11 Gene expression profiles of some of the GFP clones within 24 hour treatment with KC-L (L100) ....................................................................................................................................................... 131   xviii  List of Abbreviations  AB Acinetobacter baumannii AD Anno domini; after Christ Ag Silver Ahp Alkyl hydroperoxide reductase AIA Actinomycete isolation agar Al Aluminum As Arsenic ATCC American Type Culture Collection ATP Adenosine triphosphate B Boron Ba Barium BC Before Christ BCC Burkholderia cepacia complex BDL Below detection limit Be Beryllium BLAST Basic local alignment search tool BPY 2,2'-bipyridyl BU Buruli ulcer C Centigrade Ca Calcium Cat Catalase Cd Cadmium CF Cystic Fibrosis CFU Colony forming unit CGD Chronic granulomatous disease CLSI Clinical and Laboratory Standards Institute CM Cell/cytoplasmic membrane Co Cobalt xix Cr Chromium CRM Certified reference material Cu Cupper DFO Deferoxamine mesylate dH2O  Double deionized water DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate EDTA Ethylenediaminetetraacetic acid Eh Redox potential EM Electron microscopy ESBL Extended-spectrum β-lactamase ESKAPE Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species EtBr Ethidium bromide F Forward Fe Iron FSC Forwarded scatter channel ft Foot/feet g Gram GFP Green fluorescent protein H2O2 Hydrogen peroxide ha Hectare HCl Hydrochloric acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hg Mercury HP Hydroperoxidase HTA Hickey-Tresner agar HTS High-throughput screening ICP-MS Inductively coupled plasma mass spectrometry ICP-OES Inductively coupled plasma optical emission spectrometry In Indium  xx  ISP4 Inorganic salt starch agar K Potassium KC Kisameet clay KC-L KC aqueous leachate Kis Kisameet (vertical core samples) km Kilometer(s) KP Klebsiella pneumoniae L Liter LB Lysogeny broth/ Luria Bertani Miller LDH Layered double hydroxide Li Lithium log Logarithmic LPS Lipopolysaccharide m Meter M Molar, metal mA Milliampere MBA Metal-based antimicrobials MBC Minimum bactericidal concentration MBMM MES-buffered minimal medium MDL Minimum detection limit MDR Multidrug-resistant/Multi-drug resistance MES 2-(N-morpholino)ethanesulfonic acid mg Milligram Mg Magnesium MH Mueller-Hinton MIC Minimum inhibitory concentration  MIM Metal ion mixture min Minute(s) MIT Massachusetts Institute of Technology mL Milliliter mm Millimeter  xxi  mM Millimolar Mn Manganese Mo Molybdenum MO Metal oxide  MOH Metal hydroxide MRSA Methicillin-resistant Staphylococcus aureus mV Millivolt Na Sodium NaCl Sodium chloride NaOAc Sodium acetate NAPS Nucleic Acid Protein Service Unit NCBI National Centre for Biotechnology Information Ni Nickel NPN N-Phenylnaphthalen-1-amine NS Normal saline O Octahedral/ Oxygen OADC Oleic acid, albumin, dextrose, and catalase OD Optical density  OM Outer membrane OMT Oregon Mineral Technologies OTU Operational taxonomic unit P Phosphorous PA Pseudomonas aeruginosa Pb Lead PBS Phosphate buffered saline  PCR Polymerase chain reaction PDR Pan-drug resistant pH Power of hydrogen PI Propidium iodide ppb Parts per billion PTA Phosphotungstic acid  xxii  R Reverse RAPD Random Amplification of Polymorphic DNA RESP Respiratory RNA Ribonucleic acid ROS Reactive oxygen species rpm Revolutions per minute rRNA Ribosomal ribonucleic acid S Sulfur S8 Sulfur-8/Elemental sulfur SA Staphylococcus aureus Sb Antimony Sc Scandium Se Selenium SE Standard error sec Second(s) Si Silicon Sn Tin SOD Superoxide dismutase SPH St. Paul’s Hospital spp Species Sr Strontium SSC Side scatter channels T Tetrahedral Te Tellurium TEM Transmission Electron Microscopy/Microscope Ti Titanium Tl Thallium TLC Thin-layer chromatography TSA Tryptic soy agar TSB Tryptic soy broth U Unit  xxiii  UBC University of British Columbia USA United States of America USEPA United states Environmental Protection Agency UV Ultraviolet V Vanadium VGH Vancouver General Hospital WHO World Health Organization vol Volume wt Weight WT Wild type strain XDR Extensively-drug resistant/resistance XRD X-ray diffraction YPD Yeast extract peptone dextrose medium Zn Zinc ZOI Zone of inhibition  xxiv List of Symbols α alpha ~ around β beta ˚ degree µ micro % percent  theta  xxv  Acknowledgements       I would like to express my warm and sincere thanks to Dr. Julian Davies, my PhD supervisor, for taking me on your amazing team and for the precious guidance, inspiration, and encouragements. Julian, your special approach toward scientific research and discovery assisted me all along my PhD journey. Seeing you start the day with new research questions, sharing the media and solutions, and cracking jokes about our new findings have been a source of unforgettable joy and inspiration. I also extend my heartfelt thanks to Dr. Michael Murphy, my co-supervisor, who helped me a lot through many insightful discussion sessions. Dr. Murphy, I have learnt from you that Science is more about making good questions than seeking answers. Thank you for all your support and encouragements. I would also like to express my deep gratitude to Dr. Charles Thompson and Dr. Pauline Johnson, my committee members, for all their support and advice at committee meetings as well as fruitful comments on my thesis. My sincere appreciation goes to Dr. Vivian Miao for her continuous support. I cannot imagine my PhD studies without our discussions and her valuable inputs especially in proper scientific communication, well-established experimental design, and reviewing my posters and manuscripts. I thank Dr. Michael Gold for his cheerful conversations, having us feel we all belong to the unique family of M&I. I gratefully acknowledge the generosity of the Heiltsuk First Nation people of British Columbia for providing access to the Kisameet glacial deposit as well as Kisameet Glacial Clay Inc. for all their supports. I also acknowledge Mr. Lawrence Lund for all his supports and special efforts in collecting vertical core samples.      I would like to thank past and present members of Davies Laboratory. Thank you Ivan Villanueva for providing technical support in such an amazing and friendly atmosphere. I gratefully thank Dr. Sarah Svensson who carried out the microbiome studies in the first year and has extended her support and enthusiasm even until now from Würsburg, Germany. Dr. Mehrnoush Mohammadali, our unforgettable friendship and discussion sessions have been really heart-warming and fruitful. I appreciate Ahmad xxvi Maslati and Miguel Desmarais for their contributions in KC bacterial isolation studies. I would like to acknowledge Drs. Loretta Li, John Grace, Frank Ko, Addie Bahi, and Wanjing Xu for all our regular clay meetings and discussion sessions, enriching my understanding about clay minerals in different aspects. I am thankful to Dr. Slade loutet and Mariko Ikehata from Murphy laboratory for great discussions. I further thank Dr. James Imlay and Dr. Sergey Korshunov (University of Illinois), Dr. David Speert, Dr. James Zlosnik, and Dr. James Kronstad (UBC) for providing bacterial and fungal strains for this study. Very special thanks to Darlene Birkenhead, for her warm and caring attitude. I am very appreciative of Dr. Parvin Bolourani, Dr. Gaye Sweet, Dr. John Nomellini, Rolando Robillo, Jenny Vermeulen, and Manisha Dosanjh for being so supportive.       I am thankful for the UBC Four Year Fellowship that contributed to my academic and my professional development. I also appreciate Mitacs (UBC) that supported this study by an “Accelerate Student Fellowship Award”. Thank you Sang Mah for all your guidance and support. I would like to acknowledge that the research in this thesis was conducted at UBC, which is situated on the unceded ancestral territory of the Musqueam people. I would like to extend my deepest gratitude to my wonderful family: to my beloved father (Kayamars) and mother (Golshad), for their unconditional love and inspiration, my lovely brothers, Sirus, Reza, and Davoud, for their never-ending encouragements, and my amazing husband, Sam, for being so kind, supportive, and understanding. Very special thanks to my beloved son, Masiha, who has always been a source of love, energy, and pride to me all along this journey, especially when he tried to make me laugh with a joke to help me unwind. Masiha, I hope our exceptional journey in UBC will be a great source of inspiration and ambition for you, as my life and studies have been blessed by your precious presence.   xxvii  Dedication  This dissertation is equally dedicated to: First Nations people of Canada,  and the kind spirit of my younger sister, Sheida, who inspired me through this exceptional journey   1  Introduction and Literature Review Infections, antibiotics, and antibiotic resistance      The discovery and development of penicillin as a therapeutic agent in the early 20th century launched the golden epoch of antibiotics, which had an enormous impact on human health (Kardos and Demain 2011). The availability of antibacterial agents, once referred to as “miracle drugs”, transformed the treatment of infectious diseases and remarkably reduced morbidity and mortality caused by, formerly fatal, bacterial infections (Davies 2006; Crofts et al. 2017). Moreover, it revolutionized medicine leading to great advances in medical sciences including life-saving and life-extending procedures, critical surgeries, organ transplantation, and cancer chemotherapy (Lewis 2012; Brunel and Guery 2017). Most antibiotics were discovered by isolation and screening the chemically-diverse secondary metabolites of soil microbes (esp. Actinomycetes and fungi), as a nearly inexhaustible source of bioactive molecules during the 1940s-1960s (Fig. 1.1) (Davies 2010).        Antibiotics block or subvert key cellular functions by specific interactions with microbial targets (Fernandes 2015). Aside from the therapeutic power, mass production and global distribution caused a selective pressure and led to the spread of antimicrobial resistance two decades after their introduction (Davies and Davies 2010; McEwen and Collignon 2018). Resistance is defined as mechanisms to prevent antibiotics from hitting their targets, which includes antibiotic destruction by enzymatic modification or inactivation, target modification or overproduction, restricted penetration, and/or increased efflux of antibiotic (Davies and Davies 2010; Lewis 2013).      Although bacterial resistance mechanisms to microbial compounds are recognized as an ancient natural evolutionary phenomenon among diverse microbial ecosystems (rather than a present-day manifestation), due to the ubiquity of antibiotic production by environmental microorganisms, this feature has been artificially accelerated by the selective pressure caused by misapplication of antibacterial drugs worldwide (Wright and Poinar 2012; Lewies et al. 2018; Crofts et al. 2017). Moreover, considering the Chapter 1: Introduction and Literature Review 2 genetic flexibility of major human pathogens, rapid movement of genes within bacterial populations (both vertically and horizontally), and the enormity of the resistome (Wright 2007), rapid emergence of resistance to multiple antimicrobial agents is foreseen. Thus, fully overcoming antibiotic resistance is difficult or even impossible (Wright 2011; Allen et al. 2010). Unfortunately, no “irresistible” antibiotics are known, so the escalating emergence of multi-drug resistant (MDR) bacteria at an alarming rate together with the negligible pipeline of novel antimicrobial drugs endanger the far-reaching medical advances achieved with these therapeutic agents (Wright 2011; Ventola 2015; Frieri et al. 2016). To avoid reversion to the pre-antibiotic era, innovative and coordinated strategies toward development of new therapeutics, particularly those with novel mode of action as well as prolonging the life span and effectiveness of the current armamentarium of antibacterial agents are direly needed (Hemeg 2017; Melander and Melander 2017). Urgent need for novel antimicrobial agents       The lack of effective therapeutics for treating serious infections caused by MDR and pan-drug resistant (PDR) superbugs is fueling the urgent development of novel antimicrobial agents as well as prophylactic approaches (Spellberg and Shales 2014; Lewis 2017). Despite the alarming public health threat posed by emerging superbugs worldwide, implementation of recommended policies to manage the crisis, including sustained and coordinated antibiotic stewardship (limiting indiscriminate applications) and renewed research exploring novel therapeutics is necessary to address the problem and reduce the risks (Ventola 2015).  3 Figure 1.1 The timeline of antibiotic discovery illustrating the history of discovery and the subsequent discovery void Indicated dates represent that of the initial discovery or recorded patent. Primordial, the development of chemotherapy; golden, the period of time when most of the current conventional antibiotics were discovered; pharmacologic, more efforts toward improving the administration of antibiotics; biochemical, investigation of the biochemical aspects of antibiotic action and resistance mechanisms which together  led to chemical modification of antibiotics to circumvent  resistance; target, investigation of mode of action and relevant genetic approaches led to design of new compounds; genomic HTS, genomic sequencing techniques used to explore and predict essential  targets for incorporation into high-throughput screening studies (Silver 2011; Davies and Davies 2010; Wright 2007).Chapter 1: Introduction and Literature Review 4      Given the scientific and economic challenges facing the discovery of novel antibiotics, only two new classes of antibiotics have been introduced since the end of golden age of antibiotic discovery in the 1970s (Silver 2011). Despite innovative screening approaches and efforts to develop novel antibacterials based on genomics, high-throughput screening (HTS), combinatorial chemistry, and rational drug design, there has been a “discovery void” since 1987 (Fig. 1.1) (Lewis 2013). Since then, the majority of approved or currently developed antibiotics have been derivatives of formerly approved scaffolds against which many bacteria, expectedly, already possessed resistance mechanisms (Melander and Melander 2017). Other major scientific hurdles are the frequent rediscovery of known compounds from soil bacteria and fungi, requiring effort in dereplication to identify novel antibiotics. As well, target-focused screening of large libraries of synthetic chemical compounds has been unsuccessful partly due to poor penetration of selected compounds through the bacterial envelope (Cox et al. 2017; Lewis 2013). Furthermore, considering the history of antibiotic discovery and application, it is not unpredictable that even with a rich pipeline of novel candidates similar to our conventional antibiotics, mainly broad-spectrum single drugs; resistance will remain to be a substantial challenge to both science and medicine (Wright 2016; Spellberg et al. 2013). Thus, it is prudent to consider complementary approaches and to explore untapped natural resources, which may represent new classes of antibacterial agents with novel mechanisms as powerful underexploited weapons to combat bacterial pathogens. Alternative antimicrobial agents      Alternatives to conventional antibiotics are defined as non-compound approaches that target bacteria or the host (Czaplewski et al. 2016). Several therapeutic alternatives have gained renewed interest. Bacteriophages (both wild-type and genetically-engineered), phage lysins, antimicrobial peptides (more specifically bacteriocins produced by certain bacteria), and antibacterial nanomedicines (particularly metal-based nanoparticles) have been the most implemented approaches (Lewies 2018; Lin et al. 2017; Cotter et al. 2013; Cavalieri et al. 2014; Beyth et al. 2015). Other alternatives which mostly serve as Chapter 1: Introduction and Literature Review 5 potential adjunctive or preventive therapies include monoclonal antibodies against virulence factors or toxins of pathogens for either prophylactic or therapeutic applications (Francois et al. 2016), vaccines for new bacterial targets, quorum sensing inhibitors (quorum quenching) (Brunel and Guery 2017), probiotics to maintain or improve gut microbiota and/or to prevent colonization of pathogens, antibiofilm peptides, and immune response stimulators (Czaplewski et al. 2016; Allen et al. 2014; Nigam et al. 2014). In addition, a complementary strategy to augment the activity and longevity of current antibiotics is the use of antibiotic adjuvants, which are non-antibiotic compounds that enhance the efficacy of antibiotics through either blocking resistance, or boosting the host responses to the causative agents of infections (Wright 2016). They suppress the emergence and impact of resistance and preserve antibiotic action. Moreover, ‘’historical’’ agents such as natural clay minerals with demonstrated medicinal applications and recent studies of their antibacterial activities have been of increasing interest (Carretero 2002; Williams et al. 2004).  Natural medicinal clay minerals, their existence in human life since prehistory Traditional applications of medicinal clay minerals     Extending back into prehistoric times, natural clay minerals, one of the oldest earth materials, have been used by humans for therapeutic, nutritional, and protective purposes (Carretero et al. 2013; Gomes 2018). Ancient evidence indicates their applications for healing wounds, soothing irritations, and cleaning skin (Carretero 2002). The ancient clay tablets of Nippur (Mesopotamia, 2500 BC) referenced application of clay-based medicaments for wound treatment and hemorrhage suppression (Carretero et al. 2006). The Papyrus Eber (1600 BC) described the curative application of clay-based materials for treating certain maladies. In the ancient Greek era, Hippocrates (the father of medicine) recorded useful information about medicinal earths or terras in his writings (Gomes 2018). Nubian earth was used medicinally as an anti-inflammatory agent by ancient Egyptians, while yellow ochre (a mixture of clay and iron oxy/hydroxides) was used as a treatment for internal maladies and as a preservative in mummification Chapter 1: Introduction and Literature Review 6 (Carretero et al. 2006; Gomez and Silva 2007). In ancient Greece, mud materials were applied as antiseptics to heal skin, as cicatrices, or as a treatment for snakebites.       In some civilizations, the therapeutic application of clay materials was extended to eating the clay materials (Carretero et al. 2006). Aristotle (384-322 BC) recorded the first reference to the deliberate ingestion of earth materials (soil, clay, or ore) by humans for therapeutic purposes; this ancestral practice, nowadays called geophagy, is still in use worldwide (Abraham et al. 2010; Gomes 2018). Application of clay minerals from a therapeutic perspective as well as pharmaceutical preparations was recorded in a Roman reference dated back to 60 BC (Carretero et al. 2006). Galeno (131-201 AD), described the therapeutic application of muds to heal malaria and gastrointestinal maladies (Gomes, 2018; Carretero et al. 2006). During the Middle Ages, Avicena (980-1037 AD), the Persian physician, described and classified medicinal muds in his book “El Canon” for ingestion or topical applications; later, Ibn-al-Baitar (1197-1248 AD) disclosed curative and cosmetic applications of clay-based materials (Carretero et al. 2006). Bacci (1571) described the beneficial use of thermal mud in rheumatic pathologies, oedemas, and ulcers (Gomes 2018). During the Renaissance, clay minerals of medicinal properties were classified among other medicines in the Pharmacopeia; this coincided with the pioneering mineralogical classification, which documented the advances of mineralogy in medico-pharmaceutical matters (Carretero et al. 2006).  Modern applications for prophylactic and therapeutic purposes      In the late 19th century, a renaissance in the internal application of clay minerals led to the successful application of a clay, Bolus alba (kaolin), for treating cholera and other gasterointestinal disorders, as well as extreme septic wounds and putrid ulcers by Stumpf. His report in 1906 attracted attention for the medicinal application of kaolin and the clay was listed as a natural remedy for those maladies in pharmacopeias (Ferrell 2008; Abraham 2010; Kaolin, JAMA 1915). During the 18th-20th centuries, clay-based materials were widely employed for mud-therapy or peloid-therapy (Gomes 2018). In the 20th Chapter 1: Introduction and Literature Review  7  century, the use of healing and edible clay minerals in conventional medicine led to a readmission into pharmacopoeias. For instance, Martindale (1993) described the application of kaolin as an absorbent for the symptomatic cure of diarrhea, smectite for gastrointestinal disorders, and bentonite in the treatment of paraquat poisoning (Abraham 2010). More recently, hydrated poultices of two French green clays were applied therapeutically to treat Buruli ulcer in Ivory Coast of Africa (Williams et al. 2004).      Moreover, geophagy as an ancestral practice has been followed across the world for curative or spiritual purposes, alleviation of gastrointestinal upsets, detoxification, nutritional supplementation (due to actual physiological need, particularly Fe and Ca), or relief of famine (Young et al. 2011; Johns and Duquette 1991; Reinbacher 2003; Gomes 2013; Wilson 2003). Whilst this old practice is deeply rooted in religion and folk medicine, recent studies have reinvestigated its health benefits and the potential deleterious consequences of geophagy (Bisi-Johnson et al. 2010; Abrahams et al. 2013; Gundacker et al. 2017). Although applications of clay minerals have ancient roots, “clay science” (argillology) is a recent area of study, dating back to the mid-1930s (Bergaya and Lagaly 2013). Since then, clay sciences have significantly improved due to major advances in X-ray diffraction, electron microscopy, spectroscopy, and other analytical techniques (Zhou and Keeling 2013).  Clay and clay minerals, nanomaterials of geological origin       Natural clay minerals are the most plentiful, chemically-active components of the Earth’s surface (Velde 1995; Carretero and Lagaly 2007). They have become indispensable to modern living due to their ubiquity, having the largest number of species among inorganic materials, diverse structures and geochemical properties, which suit them for numerous technological applications, ranging from industrial materials to pharmaceutical preparations (Konta 1995; Carretero and Lagaly 2007). Clay minerals are sensitive to changing environmental conditions and their genesis happens over a variety of geological environments and throughout geological time (Rautureau et al. 2017). They are the main components of soil, sediments, weathered rocks, altered volcanic deposits, and hydrothermally-altered systems (Zhou Chapter 1: Introduction and Literature Review  8  and Keeling 2013). They are formed mostly by weathering of other silicate minerals (rock-atmosphere interface or interaction of aqueous solutions and rocks in the sedimentary piles) or by hydrothermal alteration in the late stage of magmatic cooling (Velde 1995; Zhou and Keeling 2013). In geology, clay refers to a very fine-grained (equivalent spherical diameter <2.0 μm) fraction of naturally-occurring earthy materials. Clay is composed of clay minerals as the major components, with clay-sized crystals of non-clay minerals (i.e. quartz, carbonate), metal oxides (e.g. pyrite, magnetite), and amorphous compounds (Guggenheim and Martin 1995; Bergaya and Lagaly 2013).  Clay minerals, classification and main characteristics      Natural clay minerals are composed of microcrystalline particles of hydrous charged sheet silicates (phyllosilicates) consisting of aluminum or magnesium silicates with plasticity upon hydration and hardening when fired or dried (Brigatti et al. 2006; Gomes 2018). Phyllosilicates are a major group of silicate minerals including kaolinite, smectite, micas, talc, chlorite, and other clay minerals (Fig. 1.2).       The layered structure of clay minerals is primarily composed of stable stacks of basic building blocks of tetrahedral (T) silicates [Si-O] and octahedral (O) metal oxide/hydroxide [M-O] or [M-OH] sheets, that may condense in either a 1:1 (TO, e.g. kaolinite and serpentine) or a 2:1 proportion (TOT, e.g. smectite or chlorite) (Brown 1984; Brigatti et al. 2006; Giese and Van Oss 2002) (Fig. 1.3). They possess specific physicochemical properties such as ultra-fine grain (with one dimension <2.0 μm), vast specific surface area (100’s m2/g), and high potential for reactivity (Bergaya and Lagaly 2013; Uddin 2008). Different species of clay minerals can be distinguished by their crystallo-chemical specificities (Gomes 2018). In particular, the characteristic property of hydrated clay minerals is their ion exchange capacity due to the intercalation of ions and retaining them in an exchangeable state (Konta 1995; Brigatti et al. 2006).      Clay minerals also show interlayer swelling upon hydration, high absorptive property, plasticity, colloidal capacity, and optimal rheological properties (Brigatti et al. 2006; Viseras et al. 2007). While cationic clay minerals possessing a negative charge are widespread in nature, anionic clay minerals,  Chapter 1: Introduction and Literature Review 9 referred to as layered double hydroxides (LDH) with positive charge are relatively uncommon (Ghadiri et al. 2015). LDHs are composed solely of octahedral sheets and require anions in their interlayer spaces to counterbalance their positive charge, which in turn provides them with anion exchange capacity (Choy et al. 2007; Gaskell and Hamilton 2014). Their features such as biocompatibility, tendency to intercalate various organic or inorganic anions, therapeutics, and bioactive molecules emerged them as efficient drug delivery vectors (Ladewig et al. 2009). In recent years, they have been widely investigated in the development of novel pharmaceutical formulations for wound management, cancer therapy, controlled drug delivery, and sustained drug release (Del Hoyo 2007; Gaskell and Hamilton 2014; Saifullah et al. 2014).   10 Figure 1.2 Classification of silicate minerals Clay minerals belonging to phyllosilicates shaded in blue (Bailey 1980a, b; Brown 1984; Bergaya and Lagaly 2013; Giese and Van Oss 2002).  The octahedral sheet containing trivalent metal ions (Al3+) form a di-octahedral clay mineral while octahedral sheets containing divalent metal ions (Fe2+, Mg2+) lead to the formation of a tri-octahedral clay minerals (Ghadiri et al. 2015).  Silicate examples important to this study have been bolded.  Phyllosilicates (Sheet Silicates) Silicates Tectosilicates (Framework Silicates) ● Feldspars (Albite)● Microcline● Quartz● Zeolites (Laumontite)Other Silicates Cyclosilicates (Ring Silicates) Inosilicates (Chain Silicates)Nesosilicates (Island Silicates)Sorosilicates (Couplet Silicates) 1:1Phyllosilicates Kaolinite-Serpentine 2:1Phyllosilicates 2:1 Inverted Ribbons ● Palygorskite● SepioliteKaolinite (Dioctahedral) ● Dickite● Hallosyte● Kaolinite● NacriteSerpentine (Trioctahedral) ● Amesite● Antigorite● Chrysotile● LizarditeTalc-Pyrophyllite ● Kerolite● Pimelite● TalcVermiculites Smectites Chlorites Dioctahedral Smectites ● Beidellaite● Montmorillonite● Nontronite● VolconskoiteTrioctahedral Smectites ● Hectorite● Saponite● Sauconite● SterensiteDioctahedral Chlorites ● DonbassiteTrioctahedral Chlorites ● Baileychlore● Chamosite● Clinochlore● NimiteDioctahedral Micas ● Celadonite● Illite● Muscovite● ParagoniteTrioctahedral Micas ● Biotite● Lepidolite● Phlogopite● ZinnwalditeMicas Dioctahedral Vermiculites Trioctahedral Vermiculites 11 Figure 1.3 Pharmaceutical and biomedical applications Building blocks of tetrahedral and octahedral sheets (A); 2:1 (TOT) type of phyllosilicates with interlayer exchangeable cations and water molecules e.g. smectite (B-left), 1:1 (TO) type of phyllosilicates e.g. kaolinite (B-right) (Grim 1962; An et al. 2015).  The tetrahedral sheet (T) The octahedral sheet (O) A 2:1 type phyllosilicate (TOT) 1:1 type phyllosilicate (TO) B Chapter 1: Introduction and Literature Review 12      Among nearly 4500 known minerals, about 30 have been widely employed in pharmaceutical and biomedical formulations due to their ubiquitous geological occurrence, biocompatibility, desirable physical and physicochemical properties, and low or null toxicity (Carretero and Pozo 2010; López- Galindo and Viseras 2004; Droy-Lefaix and Tateo 2006). Specific clay minerals such as smectite, kaolinite, hallosite, hectorite, saponite, palygorskite, sepiolite, and talc have been mostly used in the development of a broad range of therapeutically useful applications including pharmaceuticals, biomaterials, biosensors, cosmetics, and veterinary medicines (Ghadiri et al. 2015; Viseras et al. 2007; Gomes 2013, 2018). The fundamental physico-chemical properties of clay minerals, which can be adjusted or optimized to the intended application have been exploited for pharmaceutical applications include high surface interactions and interlayer reactions, high adsorptive property and cation exchange capacity, low hardness and abrasiveness, swelling and sorption properties, chemical inertness, optimal rheological phenomena (dispersion and viscosity), thixotropic and colloidal behaviors, thermal capacity (high heat retention), water solubility and dispersivity, plasticity features, high acid-absorbing capacity and optical attributes (Khurana et al. 2015; Choy et al. 2007; Carretero 2002). Such minerals have been employed extensively in the formulation of pharmaceutical products as active ingredients, catalysts or catalyst support excipients, or as vectors in drug delivery (Carretero and Pozo 2009, 2010; Aguzzi et al. 2007; Moraes et al. 2017; Viseras et al. 2007, 2010; Dupont and Vernisse 2009). Notably, excipients are extensively applied in pharmaceutical formulas to facilitate the preparation or promote the disintegration of pharmaceutical formulations, enhance their organoleptic characteristics (flavor corrector, pigments), improve their physicochemical features (viscosity, thickening), accelerate their elaboration (diluents, lubricants, binders, disintegrates, isotonic agents) or conservation (opacifiers, desiccants), and aid drug delivery (carrier-releasers) (Carretero and Pozo 2009; Khurana et al. 2015). They have been administered orally as gastrointestinal protectors, antacids, antidiarrhoeaics, homeostatics, osmotic oral laxatives, direct emetics, mineral supplements and antianemics, or topically as antiseptics, disinfectants, dermatological Chapter 1: Introduction and Literature Review 13 protectors, local anesthetics, and anti-inflammatories (Carretero and Pozo 2009, 2010; Ghadiri et al. 2015). In addition, clay minerals have been used in other medicinal applications including contrast diagnostic techniques due to their magnetic properties, production of dental cements in odontology, immobilization of fractures or craniofacial surgical procedures in traumatology, construction of implants and bone grafts, and aesthetic medicine (Carretero and Pozo 2009).      The beneficial applications of clay minerals for health improvement are not restricted to therapies and pharmaceutical formulas as clay has a wide scope of applications in agriculture and environmental technology, either as depolluting or detoxifying agents (Churchman et al. 2006). They have been specifically employed for removal or immobilization of harmful substances, organic pollutants, industrial wastes, and pesticides from water, or as detoxifying agents of carcinogenic mycotoxins such as aflatoxin from food crops (Nir et al. 2006; Jaynes et al. 2007; Zadaka et al. 2007). Recent studies reported efficient disinfection of water and wastewater from pathogenic microorganisms by applying sustainable clay-based materials (Unuabonah et al. 2018; Qin et al 2018). While mineral clays have been employed with favorable outcomes for centuries in pharmaceutical applications, technology, and dermopharmacy, the medical applications may have some side effects. Thus, scientific knowledge of clay properties in sufficiently controlled applications of these materials needs to be considered (Carretero et al. 2013).    Antibacterial clay minerals: a decade of studies        Despite the long history of medicinal applications of healing clay minerals, their beneficial properties in fighting infectious diseases have only recently received significant interest (Gomes and Silva 2007; Ferrell 2008). In 2002, a French humanitarian, Line Brunet de Courssou reported the successful application of hydrated French green clay poultices for healing advanced Buruli ulcer (BU) in the Ivory Coast of Africa (Brunet de Courssou 2002). BU caused by Mycobacterium ulcerans is a chronic necrotizing cutaneous ulcer found in more than 30 countries around the world, as the third most common mycobacterial disease with endemic rates in much of western and central western Africa (van Ravensway Chapter 1: Introduction and Literature Review 14 et al. 2012). However, in some African countries such as Ghana or Benin, BU has emerged to become even more prevalent than tuberculosis and/or leprosy (Portaels et al. 2009; Chany et al. 2013; Yotsu et al. 2015). The characteristic ulcers cause extensive skin loss, damage to nerves and blood vessels, and eventual deformity and disability. Despite the low mortality rate, BU has been a public-health concern in terms of morbidity, treatment, and functional disabilities (Sizaire et al. 2006).       While antibiotic therapy alone is mostly effective in the pre-ulcerative stage of this debilitating disease, with some treatments paradoxically worsening the lesions (Walsh et al. 2011; Sizaire et al. 2006; Combe et al. 2017), continued application of hydrated French green clay minerals resulted in ulcer debridement, continued tissue regeneration, and wound healing. Extended treatment with daily clay application for several months healed the ulcer with soft, supple scarring and gain of normal motor function (Brunet de Courssou 2002; Williams et al. 2004, 2008). This work brought therapeutic clay minerals into focus and led to the investigation and validation of their antibacterial activities and physico-chemical characteristics in laboratory studies (Williams and Haydel 2010).       Pioneering research reported the broad-spectrum in vitro antibacterial activity of CsAgO2 (an iron-rich illite clay from France) that was previously applied therapeutically to treat patients with BU against a broad-spectrum of bacterial pathogens including extended-spectrum β-lactamase (ESBL) Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) using growth inhibition assays (Haydel et al. 2008). Further investigations revealed that among natural healing clays worldwide, only a few deposits exhibited antibacterial properties. Despite their different mineralogical and chemical characteristics, they all originated from hydrothermally altered volcanic clastic environments, either altered pyroclastic minerals or volcanic ash, containing nanoscale expandable clay minerals and iron-rich phases (Williams et al. 2011; Morrison et al. 2016). Mpuchane et al. showed that among a total of 102 medicinal clays from South Africa, only nine exhibited antibacterial activity which was attributed to the low pH environment of the hydrated mineral suspensions, and it was further postulated that metal cations could contribute to Chapter 1: Introduction and Literature Review 15 toxicity (Mpuchane et al. 2008, 2010). Notably, recent studies have revealed that the mode(s) of action are varied among certain antibacterial clay minerals; but, may be associated with nanoparticle size, pH (either <5 or >10), redox buffering generated by hydrated clay, toxicity of specific exchanged metal ions (Fe2+,Cu2+, Al3+, and Zn2+) released from the mineral particles, or the production of antimicrobial compounds due to proliferation of resident bacterial species as reported for Jordan red soil (Williams et al. 2008, 2011; Cunningham et al. 2010; Otto and Haydel 2013a; Morrison et al. 2014, 2016; Londono and Williams 2015; Falkinham et al. 2009). For instance, Williams et al. (2011) found that the soluble clay constituents of OMT (from Oregon Mineral Technologies, Cascade Mountain) impart bactericidal properties to this clay (Williams et al. 2011).       In addition to natural antibacterial minerals, the inherent properties of some clay minerals have been modified (chemically or structurally) in order to be adjusted or optimized for the intended antibacterial applications. For instance, by chemical sorption of known bactericidal elements such as Ag, Cu, Co, and Zn onto the mineral surfaces (Bergaya and Lagaly 2001). Several studies have reported antimicrobial activities of transition metal-ion-exchanged modified clays such as allophane and imogolite (Top and Ȕlkϋ 2004; Hundáková et al. 2013; Magaña et al. 2008; Ma et al. 2010; Malachová et al. 2011; Valášková, et al. 2011), nanoparticle-exchanged clays (Su et al. 2011), and synthetic clays (Parolo et al. 2011; Gaskell and Hamilton 2014).   Kisameet clay, history of therapeutic applications      Kisameet clay (KC), formerly known as Canadian Canamin, B.C. Peloid deposit, Namu clay, or Canadian natural clay, is a large nonmarine deposit of clay mineral found on the north shore of Kisameet Bay (51°58’20”N/127°52’50”W). It is located at an elevation of 15 meters above sea level near the southern tip of King Island, 7.5 miles north of the Namu community, 55 miles south west of Bella Coola, and around 280 miles northwest of Vancouver on the Central Coast of British Columbia, Canada (Fig. 1.4A) (Minister of Mines 1952; British Columbia Geological Survey 2011). Kisameet clay is a very fine-Chapter 1: Introduction and Literature Review 16 grained glacial clay mineral at the bottom of a small depression about 400 ft inland and around 50 ft above the shoreline of Kisameet Bay. The clay presumably is Quaternary in age, resulting from glacial expansion and retreat during past ice ages, but lies upon black and gray gneiss and schist of the Jurassic to Tertiary Coast Plutonic Complex (Minister of Mines 1952; Svensson et al. 2017).           The local Heiltsuk First Nation people have exploited the exceptional healing properties of this natural clay for treating various types of skin irritations and internal maladies for generations (Hauser 1950, 1952). The KC deposit remains mostly untouched, but it was drilled in 1946 for the initial investigation in which the dimensions of the deposit were established by around 77 vertical and inclined drill holes, and samples were submitted to the British Columbia Mines Branch (Minister of Mines 1952; Geological Survey of Canada Map 1386A). As shown by drill logs in Fig. 1.4B, the deposit covers an area around 2 ha (5.2 acre), with an estimated 181,000 tons of clay mineral in a depression underlain by sand, gravel, and bedrocks to a range of thickness from 1- 42 ft (Minister of Mines 1952). The clay was reported to be of plutonic and volcanic origin, with very fine grain size and remarkably uniform texture (Hauser and Colombo 1953). Geological studies with aerial mapping indicated the presence of acid plutonic rocks to the north on King Island. Systematic surveys revealed that the deposit is overlain by a 1-6 ft layer of overburden, mostly humus and organic materials (Minister of Mines 1952; Hauser 1952). KC is naturally found as thick paste or viscous liquid with a soft putty-like consistency as mined (Hauser 1952). It is dark blue-grey when moist and pale grey when dry.     Later, KC was sold in a water-suspension (Absor-Vite) to be administered orally or in jars as mud (Ray-Vite) for topical applications (Ure et al. 1946). Successful oral application of Absor-Vite (faintly alkaline) for treatment of gastric ulcer, stomach distress, duodenal ulcer, and intestinal disorders such as ulcerative colitis has been described by Ure et al. Further X-ray examinations confirmed the treatment of gastric and duodenal ulcers previously seen on X-rays. The healing action in clinical applications was attributed to the remarkable buffering action and neutralizing action of acid in stomach, and formation of Chapter 1: Introduction and Literature Review 17 a soothing and protective film, covering the mucosa of the gastric ulcers and ulcer crater (Ure et al. 1946). Moreover, topical applications of Ray-Vite for healing arthritis and neuritis, sciatica, varicose ulcer, phlebitis, eczema, skin irritations (particularly athlete’s foot), wound and burns (even third-degree) have been described (Hauser 1953). These external applications suggested that KC preparations harbor antiphlogistic and anesthetic properties, and intriguingly antibacterial activities (Ure et al. 1946). Furthermore, in the veterinary field, successful applications of KC for treating scours, milk fever, acetonemia (ketosis), and other ailments have been recorded (Hauser 1952). 18 Figure 1.4 The Kisameet Bay glacial clay deposit The approximate location in the map of central coast of British Columbia, Canada (A); The KC deposit map, modified from Minister of Mines1952 (B). A B Chapter 1: Introduction and Literature Review 19      The first systematic mineralogical analyses and electrophoretic studies of KC deposit conducted by Ernst Hauser revealed that KC consisted mainly of negatively charged ultrafine particles of highly colloidal aluminum silicate (with calcium ions as counter ions) (Hauser 1952).  Further thermal analysis showed that KC drastically differs from bentonite, but with a close similarity to standard silica gels and quartz (Hauser 1952). X-ray diffraction studies also revealed that aside from the silica layers, this aluminum silicate differed from other known clay minerals (at that time) in terms of its composition and properties. Further ultramicroscopy and electron micrograph studies showed that KC is not of uniform composition and demonstrated its heterogeneity by the presence of mica-type particles and comparatively large silica particles (Hauser 1952, 1955). Initial chemical analysis indicated that KC is an iron-rich clay (8.3% wt/wt iron oxides and 19.4% alumina) which is fine-grained (54.6% with a grain diameter of 1.7 to 3 µm and 30.7% with a diameter of <1.7 µm). Part of the remaining coarser fraction consists of fresh-looking mineral fragments, primarily quartz, biotite, feldspar, and hornblende (Hauser 1952; Ure et al. 1946). KC exhibited marked buffering action and neutralizing properties and another feature which clearly differentiates this clay from other known minerals was the structure of films obtained by spreading the dispersion on a flat surface (Ure et al. 1946).   Antibacterial activity reported in 1946      In 1941, Dr. Duff (UBC) investigated the antibacterial properties of Ray-Vite by addition of clay suspension to broth cultures of E. coli and S. aureus and reported that while a large dose of clay inhibited bacterial growth, small doses causeed poor and slow growth of treated organisms (Ure et al., 1946). The inhibitory action was attributed to the adsorptive action of clay. Further experiments comparing the activity to that of charcoal or another clay suggested that KC (Ray-Vite) possessed antibacterial activity, which was not found in charcoal and by another clay in addition to the adsorptive property found in other clay minerals. However, in 1951, investigation of the antibacterial activity of clay at the Massachusetts Institute of Technology (MIT) demonstrated no inhibitory action on the growth of E. coli, S. aureus, or Chapter 1: Introduction and Literature Review 20 Bacillus species (Maurer 1951). It was ascertained that the superfine and fine fractions of KC deposit exhibited antibacterial properties, which however masked to a respectively high degree in the natural and even screened products (Hauser 1953). Collectively, based on the successful applications of KC for medical/human and veterinary professions, Hauser emphasized that it deserved specific attention from mineralogical aspects and colloid chemistry. In addition, further investigations of its geological properties from a medical point of view as well as systematic purification of clay based on the colloidal properties were recommended (Hauser 1952, 1953).  Research objectives      As there are some conflicting records on the antibacterial activity of KC (natural and/or commercially available products in 1940s-1950s), the primary goal of this research was to confirm and investigate the antimicrobial properties of KC samples and further, to identify the spectrum of the activity by testing a variety of bacterial and fungal human pathogens. Despite anecdotal accounts of KC’s medicinal qualities together with reported clinical observations, there is limited information concerning its chemical, biological, and physical composition with respect to its antibacterial properties. With a series of integrated microbiological, mineralogical, and chemical studies, this research focuses on the characterization of the physicochemical and/or biological properties of KC that may contribute to its antibacterial activity. Moreover, as natural clay minerals are heterogeneous products due to their complex and diverse composition, continuously affected by environmental factors, this research provides support for exploring the mode(s) of action and defining the active principal components of KC. These results should provide a better understanding of KC properties related to its antibacterial activity and will be vital in further efforts in order to develop the formulation of a consistent preparation to expand the therapeutic possibilities of KC as a natural mineral-based antimicrobial agent.  21  Characterization of Kisameet Clay Introduction     Since there is limited general knowledge on KC properties, mostly obtained from processed samples in the 1950s, the goal of this chapter is to provide the first comprehensive characterization of the Kisameet glacial clay deposit in terms of its geochemical, mineralogical, and biological properties, by investigating a variety of KC samples collected from different locations in the deposit. This provides a framework for a better understanding of the basis for the unique properties of KC including its antibacterial action and the geochemical variables that may influence this activity.  Materials and methods Clay sample collection and preparation      The unprocessed KC samples used in this study were supplied by Kisameet Glacial Clay Inc. in the original wet form. Bucket samples (KC14, KC35) were collected two years prior to testing from 0-1 ft depth from Kisameet Bay (Fig. 1.4B) and stored at room temperature in sealed buckets. Five vertical cores (Kis1 to Kis5) were harvested from five different locations in Kisameet Bay on 24 October 2012 (Fig. 2.1). As the depth of the deposit is variable, Kis1 extended from 0 ft to ~32 ft, while the bottom of the Kis4 core was only ~20 ft below the surface. Each core was then divided into 4-ft-long sections and labeled according to the depth at the top of the section. For instance, the Kis1 core was separated into samples Kis1-0 (0 ft ≤ depth <4 ft), Kis1-4 (4 ft ≤ depth < 8 ft), Kis1-8 (8 ft ≤ depth <12 ft), and so on, where the first and second numbers indicate the core and the depth, respectively. Vertical cores were transported to UBC and stored sealed, in a vertical position in a cold room at 4˚C in the dark under normal atmospheric conditions. Following scientific records and labeling, the cores were opened and the top 10 cm from each 4 ft length core was collected for physicochemical analyses and biotic characterization. To avoid cross-mixture between different depths within a core, only the interior Chapter 2: Characterization of Kisameet Clay 22 undisturbed part (fraction) of each core was used for further analyses. Clay samples were dried in a vacuum desiccator at room temperature or by heating in an oven at 60˚C. Dry KC samples were ground by mortar and pestle and autoclaved at 121˚C for 1 h before experimental analyses. Figure 2.1 Map of the Kisameet Bay glacial clay deposit, spatial description of vertical core sampling The approximate location of Kisameet Bay on the central coast of British Columbia around 450 km northwest of Vancouver, Canada (top right inset). The topographical layout of the deposit providing the approximate locations of the five vertical core samples (Kis1 to Kis5) and bucket sample KC35. The KC deposit map has been modified/simplified from Minister of Mines, Province of British Columbia, 1952; (Svensson et al. 2017). Chapter 2: Characterization of Kisameet Clay  23   Measurement of pH and redox potential      Measurements of pH and redox potential were performed based on standard methods. pH measurement of KC was performed using either equilibrated suspensions of 1 g clay in 10 mL deionized water (dH2O) or aqueous leachates using a VWR-SB20 pH meter. The redox potentials were measured using a Beckman Phi44 pH meter with means for temperature compensation (with redox probe) (USEPA method 9045D, 2007).   Mineralogical composition by X-ray diffraction      Quantitative mineralogical profiles of KC clay samples were analyzed by X-ray diffraction (Bish 1993). The clay samples were reduced to the optimum grain-size range (<10 m) for quantitative X-ray analysis by grinding under ethanol in a vibratory McCrone Micronising Mill for 7 minutes. Continuous-scan X-ray powder-diffraction data were obtained over a range 3-80° 2-  range with step-size increments of 0.03° 2- and a counting time of 0.7 second per step, with CoKalpha radiation on a Bruker D8 Focus Bragg-Brentano diffractometer equipped with an Fe monochromator foil, 0.6 mm (0.3°) divergence slit, incident- and diffracted-beam Soller slits, and a LynxEye detector. The long fine-focus Co X-ray tube was operated at 35 kV and 40 mA, using a take-off angle of 6°. The X-ray diffractograms were analyzed using the International Centre for Diffraction Database PDF-4 and Search-Match software by Bruker. X-ray powder-diffraction data of the clay samples were refined with Rietveld program Topas 4.2 (Bruker AXS).  Aqueous leachate preparation      Aqueous leachates were prepared from 5% (wt/vol) suspensions of the original clay in dH2O with a pH ~6.9. The water content of each clay sample was determined by measuring the mass lost after heat-drying of one gram of original clay samples. One gram of wet clay was weighed and suspended in sterile dH2O for a final concentration of 5% (~20 mL) in an sterile flask, and incubated at room temperature with shaking at 200 revolutions per minute (rpm) for 24 h. Suspensions were then centrifuged at 20,000 rpm Chapter 2: Characterization of Kisameet Clay  24  for 2 h to remove particulate matter. Leachates were further clarified by filtration of the supernatant through a 0.22 m filter (Millipore). The resulting aqueous leachates were stored sealed in the dark at 4°C until testing.  Elemental analysis of bulk clay samples and aqueous leachates by inductively coupled plasma optical emission spectrometry (ICP-OES)       The elemental composition of bulk KC samples were determined using ICP-OES after acid digestion according to USEPA method 3050B (USEPA 2007). Briefly, one gram of oven-dried sample of each clay was digested with repeated additions of concentrated nitric acid and 30% hydrogen peroxide. Concentrated hydrochloric acid was added to the initial digestate and the sample was refluxed. The digestate was filtered through a Whatman No. 41 filter paper and residues were rinsed with hot hydrochloric acid (HCl) and then hot water. Filter paper and other residues were returned to the digestion flask, refluxed with additional HCl and then filtered again. The digestate was then diluted to a final volume of 100 mL for ICP-OES analysis (USEPA 2007). The detection limits of ICP-OES have been recorded in Table 2.3.   Antibacterial activity of vertical core samples      Quantification of antibacterial activity was performed using E. coli K-12 MG1655 (Bachmann 1972) as a test strain in a viability assay. Briefly, cultures of E. coli MG1655 in Luria-Bertani Miller broth (LB) were diluted into the fresh LB to an approximate concentration of ~107 CFU (colony forming units) mL-1 aerobically and incubated at 37˚C with gentle rotary mixing at 200 rpm to reach exponential phase of growth. Ten milligrams of dried autoclaved clay samples were suspended in 1 mL of sterile dH2O. Exponential-phase cultures of E. coli (OD600 ~0.4) were washed twice with sterile dH2O, and used to inoculate 1% clay suspensions tubes with approximately 107 CFU mL-1 of bacteria. Clay-bacteria suspensions were incubated with gentle rotary shaking at 37°C for 24 h to prevent sedimentation, serially diluted and plated on LB agar for CFU enumeration. Sterile water was used as a negative control. To Chapter 2: Characterization of Kisameet Clay 25 control for the low pH of some KC suspensions, the viability of E. coli in 100 mM phosphate buffer, pH 4.3 (the pH of KC35 aqueous leachate) was determined. Isolation of KC resident bacteria using different selective and differential media     Viable bacteria from cores (Kis2, Kis3, Kis5) and buckets (KC14, KC35) were cultured from wet unautoclaved clay samples stored under natural atmospheric conditions at 4˚C. Approximately 100 mg of wet clay samples of Kis2 (32 ft), Kis3-0, and Kis5 (4 to 28 ft), KC14, and KC35 were suspended in 1 mL sterile normal saline (NS) (0.85% wt/vol) and vortexed and homogenized. Subsequently, those original 10% suspensions were diluted serially in sterile NS to make 1% and 0.1% (wt/vol) dilutions. 200 µl of serially diluted KC suspensions were plated on either agar media including Hickey-Tresner agar (HTA), Actinomycete isolation agar (AIA), ISP4, or M1 media (all supplemented with 20 µg mL-1 benomyl as an antifungal agent) or in broth media including Tryptic soy broth (TSB), OA medium, 1% (wt/vol) N-Z amine (Sigma), and plant peptones (lupin, pea, potato, soy) (Solabia). All liquid cultures were incubated at 30˚C with gentle shaking; after one week, 200 µl of each broth culture was plated on the same media supplemented with 1.5% agar and incubated for 1-4 weeks at 30˚C. Colonies with different morphologies were picked aseptically and streaked on fresh agar media to obtain pure isolated colonies for further identification. (More details about the media recipes and preparation in Appendix A.1). Identification of KC bacterial isolates by 16S rRNA sequencing      KC bacterial isolates were identified using 16S rRNA gene sequencing as the most suitable gene target for investigating bacterial phylogeny and diversity (Weisburg et al. 1991). Colony lysates were used as template DNA for Polymerase chain reactions (PCR). Briefly, one or two pure colonies were mixed well in 100 μL of sterile dH2O and the tubes were heated in a PCR machine at 99.9˚C for 10 mins. A PCR reaction master mix composed of 4 μL of reaction buffer-Mg2+ (10X), 1.2 μL of 50 mM MgCl2, 0.9 μL of dNTP mixture (10 mM), 0.2 μL of Taq polymerase (5 U/μL), 1.5 μL of each primer (10 μM) and 25.7 μL of nuclease-free sterile dH2O. The reaction mixture was mixed with 1 μL of template DNA.  Chapter 2: Characterization of Kisameet Clay  26       PCR amplification was performed using a 2720 Thermal Cycler (Applied Biosystems). The PCR program with two universal primers (27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGGYTACCTTGTTACGACTT-3') was as follows: initial denaturation (96˚C, 2 min), denaturation (94˚C, 15 sec); annealing (55˚C 15 sec); elongation (72˚C, 90 sec); final extension (72˚C, 10 min) for 35 cycles. Products of PCR amplification were analyzed by gel electrophoresis (in 1% (wt/vol) agarose in 0.5x TAE buffer)) stained with ethidium bromide (EtBr) and visualized using a UV transilluminator and Light box. PCR products were cleaned up following the isopropanol precipitation procedure before sequencing. The PCR products were first mixed with 3M sodium acetate (NaOAc) (0.1x PCR product volume) and isopropanol (0.7x PCR product volume), vortexed briefly and centrifuged for 10 min at 14,000 rpm at room temperature. After gentle decanting of the supernatant, the pellet was resuspended in 200 μL of 70% ethanol to remove the remaining salt. The solution was centrifuged for 2-3 min at 14,000 rpm and the pellet was vacuum-dried in a Speed Vac and resuspended in 25 μL of nuclease free sterile water. The concentration of nucleic acids in 16S rRNA gene amplified products were measured using microspectrophotometery (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, Delaware, USA). Absorbance was measured at wavelengths of 260 (A260) and 280 (A280) nm. DNA with absorbance quotient (A260/A280) between 1.8 and 2.0 was considered purified. Purified products were sent to either Genewiz (South Plainfield, New Jersey, USA) or the Nucleic Acid Protein Service Unit (NAPS UBC, Vancouver, Canada), or Macrogen (Geumcheon-gu, Seoul, Korea) for DNA sequencing. Sequencing data were analyzed using BioNumerics Software (v.5.10). Basic local alignment tool (BLAST) was used for rapid comparison of nucleotide sequences and the phylogenetic tree of KC isolates was assembled based on V2, V3, and V4 regions of 16S rRNA gene sequences by the neighbour joining method in macVector (v.13.0). Genetic distances were generated using the Kimura 2-parameter method. 16S rRNA gene sequence of E. coli K12 was used as a reference. Chapter 2: Characterization of Kisameet Clay  27   Results  KC vertical cores and bucket samples      Five vertical cores (Kis1 to Kis5) were collected from the deposit at five different locations (Fig. 2.1). As each core was divided into four-feet-long sections, in total 29 of these sections in addition to the KC35 bucket sample were selected for physicochemical characterizations. The shallow core samples (depth 0-4 ft), consisted primarily of organic materials (Kis 1, Kis 2, Kis 3, Kis 5) were separated into “organic” (-0a) and “clay” (-0) fractions. The Kisameet glacial clay deposit is covered by an organic overburden and humus layer of varying thickness as shown in Fig. 2.2A as it is present in some shallow (i.e. 0-4 ft) vertical core samples (Fig. 2.2B). The clay is originally of paste-like consistency, with mostly dark greenish-gray color as mined in its original moist form and light gray when dry (Fig. 2.2D). Areas of reddish-brown material can also be present, especially upon exposure to air as seen at the surface of bucket samples (Fig. 2.2C).  KC is very fine grained and feels smooth and sticky in its natural state, while the presence of coarse particles is revealed by rubbing on the skin as reported previously (Hauser 1952). Light-microscopy studies of KC aqueous suspensions revealed the presence of comparatively large silica and mica-type particles as previously reported (Hauser and Colombo 1953; Hauser 1955) (Fig. 2.3).              28     A layer of organic overburden and humus of varying thickness covered the Kisameet deposit (A); Kis5 tubes as examples of KC vertical core samples presenting the green-gray color of the clay in situ, as well as organic overburden present in some shallow (i.e. 0-4 ft core) samples from the top of deposit (B); KC35 bucket sample, dark greenish clay with areas of reddish-brown material upon exposure to air (C); KC sample following exposure to air, showing reddish-brown area (left), as well as dried and powdered sample of clay (right) (D). A C D B Figure 2.2 Examples of Kisameet clay samples Kis5-4 Kis5-8 Kis5-0 5 cm Chapter 2: Characterization of Kisameet Clay 29 Figure 2.3 Light microscopy photographs of KC particles, presence of large silica and mica-type particles (X400) Mineralogical compositions      The results of quantitative phase analysis by Rietveld refinements are summarized in Table 2.1. KC can be classified as a mixture of framework silicates and illite/chlorite type phyllosilicates, composed of silicate minerals (85.8-100%), mainly of tectosilicates (53.8-79.2%) known as framework silicates. KC contains phyllosilicates (sheet silicates) (10.8-36.6%) including biotite (3.3-17.1%), illite-type mica (2.3-9.7%), and chlorite-type clinochlore (5.2-13.3%). KC samples also contain 6.8-22.5% quartz, and 8.9-16.3% actinolite, which is an inosilicate mineral. Collectively, mineralogical analysis revealed no major differences between the selected core samples and KC35. Likewise, there was no great variation among different KC core samples, except for Kis5-0, which contained 4.4% goethite, Kis3-28 with 1.1% magnetite, as iron hydroxide/oxides, and also the presence of calcite in Kis5 4-24 ft samples (0.3-0.4%). Physicochemical properties of KC samples including pH, redox, and elemental analyses      Physical and chemical properties of clay samples including their water content, color, pH and redox potential have been summarized in Table 2.2. Water content was found to be between 28.3-60.3 % for all Chapter 2: Characterization of Kisameet Clay 30 clay samples, while organic surface sample (-0a) of Kis1, Kis2, Kis3, and Kis5, composed mostly of organic material had 62.5-83.9% moisture content. These observations revealed that deeper KC samples tend to have a darker gray color. Comparison of the pH of core sample suspensions revealed marked variations based on depth (Fig. 2.4A). While previous studies suggested that KC is mildly alkaline (pH ~8) (Hauser 1952, Williams et al. 2011), the low pH of KC35 and some core samples from surface of deposit suggests that some KC samples could be markedly more acidic. The KC deposit has a significant organic overburden (Fig. 2.2A), which might decrease the pH at the surface. Moreover, while the pH of prepared aqueous leachates showed the same positive correlation between increasing pH and depth, we noted that aqueous leachates generally showed a lower pH than their suspension counterparts (Fig. 2.4A, B). This may reflect a lowering of pH during the 24 h of incubation under aeration during leachate preparation. In support of this, remeasurement of suspension pHs a few months after core cutting resulted in markedly higher levels of acidity. Moreover, the pH of KC35, which was harvested at least 2 years before the core samples and stored at 4°C, had comparatively lower suspension pH of 4.3 (Table 2.2 and Fig. 2.4A). In addition, measurement of different preparations of KC core samples (fresh aqueous suspensions, aqueous leachates, or suspensions of clay that had been stored exposed to air for approximately 1 month) suggested that the clay pH could vary depending on (i) depth of the sample in the deposit and (ii) exposure to air. First, the pH of fresh KC suspensions revealed a strong trend with depth (Table 2.2 and Fig. 2.4A), with the pH of clay from greater depths significantly higher than for samples from depths of 0 to 8 ft (pH 9.5 versus pH 8.0; p < 0.0001). As speciation, different chemical forms, could presumably, affect the antibacterial activity of toxic metals, including Fe, the redox potential of KC samples was also determined (Table 2.2, Fig. 2.4C). The measured redox potential of the core samples varied relatively widely from +19 mV to a maximum of +373 mV, with the redox potential of KC35 being +427 mV. Chemical analysis of acid-digested elements in KC core samples demonstrated no major variations among different KC samples but confirmed previous observations of significant amounts of Fe and Al (~104 mg/kg). Values for elemental analyses of bulk clay are summarized in Table 2.3. 31 Table 2.1 Mineralogical composition of selected KC core samples The percent of each indicated mineral was determined by XRD.  These amounts represent the relative amounts of crystalline phases normalized to 100% amorphous-free. Mineral Classification Ideal Formula  KC35  1-0*  1-12  1-24  2-0*  2-36  3-0*  3-12  3-28  4-0*  4-16  5-0  5-4  5-8  5-12  5-16  5-20  5-24  5-28 Actinolite Inosilicate Ca2(Mg,Fe2+)5Si8O22(OH)2 9.3 9.9 10.8 10.0 10.1 9.9 16.3 9.5 8.9 10.6 10.9 nd 10.1 9.9 9.3 9.3 9.7 9.1 9.8 Albite low, calcian Tectosilicate NaAlSi3O8 40.8 39.0 37.7 37.8 38.6 43.3 39.6 36.2 44.7 40.3 38.4 42.0 37.3 39.3 36.5 36.6 35.5 35.2 38.3 Biotite 1M Phyllosilicate K(Mg,Fe)3AlSi3O10(OH)2 10.0 11.5 13.6 14.4 11.7 5.9 8.8 15.5 3.3 10.6 14.0 11.1 13.9 12.1 16.2 16.1 17.1 16.6 14.5 Calcite Calcium carbonate CaCO3 nd** nd nd nd nd nd nd nd nd nd nd nd 0.3 0.3 0.4 0.4 0.3 0.4 nd Clinochlore II Phyllosilicate (Mg,Fe2+)5Al(Si3Al)O10(OH)8 9.0 10.3 11.4 12.7 11.0 7.6 9.4 13.3 5.2 8.5 9.7 nd 8.7 8.1 9.6 9.6 10.1 10.3 11.6 Goethite Iron-bearing hydroxide FeO(OH) nd nd nd nd nd nd nd nd nd nd nd 4.4 nd nd nd nd nd nd nd Gypsum Soft sulphate CaSO4·2H2O 0.9 0.7 nd nd nd 0.3 nd 0.2 nd nd nd 9.8 nd nd nd nd nd nd nd Illite-Muscovite Phyllosilicate K0.65Al2.0Al0.65Si3.35O10(OH)2 5.3 5.2 5.5 5.8 5.4 4.6 4.5 5.2 2.3 5.2 5.7 nd 8.3 6.7 8.0 7.5 9.0 9.7 5.9 Laumontite Tectosilicate CaAl2Si4O12·4H2O 3.5 4.1 3.5 3.1 4.1 1.9 3.0 4.4 2.0 3.8 3.4 nd 3.8 3.6 3.4 3.5 3.1 2.9 3.4 Magnetite Iron oxide Fe3O4 nd nd nd nd nd nd nd nd 1.1 nd nd nd nd nd nd nd nd nd nd Microcline ordered Tectosilicate KAlSi3O8 8.9 8.7 9.1 8.6 9.0 8.9 8.2 8.8 10.0 9.5 9.4 11.6 8.5 9.5 9.0 9.2 8.0 9.0 8.9 Quartz low Tectosilicate SiO2 12.2 10.5 8.5 7.7 10.0 17.5 10.2 6.9 22.5 11.5 8.5 21.1 9.1 10.4 7.6 7.8 7.2 6.8 7.6 Total tectosilicates 65.4 62.3 58.8 57.2 61.7 71.6 61.0 56.3 79.2 65.1 59.7 74.7 58.7 62.8 56.5 57.1 53.8 53.9 58.2 Total phyllosilicates 24.3 27.0 30.5 32.9 28.1 18.1 22.7 34.0 10.8 24.3 29.4 11.1 30.9 26.9 33.8 33.2 36.2 36.6 32.0 Total inosilicate 9.3 9.9 10.8 10.0 10.1 9.9 16.3 9.5 8.9 10.6 10.9 nd 10.1 9.9 9.3 9.3 9.7 9.1 9.8 Total silicates 96.7 99.2 100.0 100.0 99.9 99.6 100.0 99.8 98.9 100.0 100.0 85.8 99.7 99.6 99.6 99.6 99.7 99.6 100.0 Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0* 0-4 ft blend bulk sample** not detected32 Table 2.2 Physicochemical properties of KC samples based on depth The scale of clay color darkness is on the right. KC35 1-0a 1-0 1-4 1-8 1-12  1-16  1-20  1-24  1-28 2-0a 2-0 2-36 3-0a 3-0 3-4Water content (% Wt) 31.5 78.1 34.8 32.2 40.6 45.1 40.6 42.8 42.9 39.6 38.5 28.3 28.3 62.5 38.2 30.7 color Gray Brown  Gray Gray Gray Gray Gray Gray Gray Gray Brown Gray Gray Brown Gray Gray Darkness 2 na* 1  1-2 1-1.5  1-1.5 2 2 3 3.5-4 na 1  3-4 na 1 1-1.5 pH (aqueous suspension) 4.30 4.73 6.90 7.21 8.84 9.47 9.67 9.76 9.61 9.50 8.28 7.44 9.31 6.86 8.04 8.35 pH (aqueous leachate) 4.35 3.97 4.71 7.56 7.34 7.50 7.43 7.59 7.34 6.81 4.90 7.44 6.91 5.40 6.14 6.45 Redox, Eh (mV) 427.0 273.0 286.0 213.0 69.8 19.0 161.9 60.3 104.4 209.0 212.0 293.0 202.0 211.0 74.6 76.8 11.53-8 3-12  3-16  3-20  3-24  3-28 4-0 4-16 5-0a 5-4 5-8 5-12  5-16  5-20  5-24  5-28 2Water content (% Wt) 33.1 38.9 37.9 42.9 41.2 39.6 40.3 41.7 83.9 45.9 40.4 60.3 55.9 46.5 43.6 45.8 2.5color Gray Gray Gray Gray Gray Gray Brown Gray Brown Gray Gray Gray Gray Gray Gray Gray 3Darkness 1 1-1.5 1.5-2 2-2.5 3.5-5 5 na  3-4 na 2 1.5-2 2 2  2-3 3 4 3.5pH (aqueous suspension) 9.07 9.33 9.61 9.43 9.57 9.48 8.50 9.66 7.75 7.38 7.51 8.21 8.84 9.26 9.34 9.50 4pH (aqueous leachate) 6.80 6.90 7.11 7.06 7.13 7.03 7.61 7.76 4.32 5.86 6.27 6.74 6.91 6.92 6.95 7.54 4.5Redox, Eh (mV) 122.2 75.6 120.4 46.8 53.8 130.5 201.0 171.5 373.0 nd nd** nd nd nd nd 201.0 5* na, not applicable** nd, not determined33 Table 2.3 Acid-digested elemental composition of KC bulk clay samples Analyste levels were determined by ICP-OES and are reported in mg/kg. MDL: minimum detection limit (mg/kg) Analyte KC35  1-0  1-4  1-8  1-12  1-16  1-20  1-24  1-28  2-0 2-36  3-0  3-4  3-8 Emission (nm) MDL Ag BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 328.068 0.0024 Al 4.70E+04 4.25E+04 3.68E+04 2.91E+04 2.80E+04 3.72E+04 3.18E+04 3.34E+04 3.35E+04 2.51E+04 3.20E+04 3.46E+03 3.63E+04 3.58E+04 396.153 0.0083 As BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 188.979 0.0361 Ba 5.61E+02 4.90E+02 4.52E+02 3.67E+02 3.88E+02 4.78E+02 4.17E+02 4.42E+02 3.89E+02 2.84E+02 3.36E+02 1.39E+01 4.27E+02 4.83E+02 233.527 0.0006 Be 3.41E+00 3.11E+00 2.87E+00 1.99E+00 2.06E+00 2.35E+00 2.16E+00 2.29E+00 2.31E+00 1.87E+00 1.98E+00 BDL 2.81E+00 2.37E+00 313.107 0.0005 Ca 1.42E+04 1.25E+04 1.14E+04 7.53E+03 6.79E+03 8.47E+03 6.89E+03 7.99E+03 9.99E+03 6.77E+03 1.33E+04 1.27E+03 1.13E+04 1.14E+04 317.933 0.01 Cd 1.92E+00 3.16E+00 2.26E+00 BDL 2.66E+00 BDL BDL 5.51E-01 2.81E+00 BDL 2.98E+00 6.01E+00 2.45E+00 BDL 228.802 0.0019 Co 4.02E+01 3.54E+01 3.42E+01 2.45E+01 2.56E+01 3.05E+01 2.69E+01 2.88E+01 2.87E+01 2.38E+01 3.01E+01 1.32E+00 3.34E+01 3.31E+01 228.616 0.0026 Cr 3.37E+01 2.89E+01 3.00E+01 2.11E+01 2.35E+01 2.21E+01 2.05E+01 2.57E+01 2.41E+01 2.06E+01 4.04E+01 1.26E+01 2.90E+01 2.68E+01 267.716 0.0011 Cu 1.76E+02 1.08E+02 7.40E+02 1.21E+02 1.02E+02 8.00E+01 8.16E+01 8.31E+01 1.11E+02 1.11E+02 1.83E+02 1.17E+02 1.13E+02 1.37E+02 327.393 0.0042 Fe 3.32E+04 2.02E+04 4.32E+04 1.11E+04 2.81E+04 9.36E+03 1.45E+04 1.56E+04 1.94E+04 2.84E+04 3.23E+04 1.33E+03 2.32E+04 3.86E+04 238.204 0.0077 K 1.83E+04 1.57E+04 1.56E+04 1.14E+04 1.31E+04 1.39E+04 1.33E+04 1.42E+04 1.38E+04 9.81E+03 1.17E+04 5.99E+02 1.45E+04 1.63E+04 766.49 0.05 Mg 2.88E+04 2.51E+04 2.45E+04 1.70E+04 1.89E+04 2.03E+04 1.95E+04 2.05E+04 2.10E+04 1.56E+04 2.22E+04 5.63E+02 2.22E+04 2.38E+04 285.213 0.6521 Mn 1.47E+03 1.28E+03 1.22E+03 9.49E+02 9.99E+02 1.20E+03 1.05E+03 1.09E+03 1.09E+03 7.66E+02 1.10E+03 2.36E+01 1.13E+03 1.22E+03 257.61 0.0136 Mo 1.31E+00 6.25E-01 1.44E+00 BDL BDL BDL BDL 2.78E-01 3.43E-01 2.06E+00 6.02E+00 1.69E+00 1.11E+00 BDL 202.031 0.0058 Na 1.40E+03 1.12E+03 1.27E+03 1.38E+03 1.86E+03 2.28E+03 1.71E+03 1.35E+03 1.12E+03 8.74E+02 9.90E+02 BDL 1.20E+03 1.04E+03 589.592 0.05 Ni 2.96E+01 2.49E+01 2.65E+01 1.83E+01 1.91E+01 1.99E+01 1.83E+01 2.20E+01 2.35E+01 1.96E+01 3.50E+01 6.39E+00 2.54E+01 2.39E+01 231.604 0.004 P 1.52E+03 1.46E+03 1.38E+03 9.65E+02 8.07E+02 1.01E+03 7.50E+02 7.42E+02 8.46E+02 9.57E+02 2.10E+03 1.80E+02 1.28E+03 1.92E+03 213.617 0.05 Pb 3.19E+01 2.31E+01 5.05E+01 2.04E+01 1.71E+01 8.75E+00 9.87E+00 1.04E+01 1.48E+01 1.69E+01 3.56E+01 3.53E+01 4.10E+01 5.04E+02 220.353 0.0029 S 9.54E+03 8.84E+03 2.24E+03 1.24E+03 7.95E+02 8.97E+02 3.52E+02 5.93E+02 1.12E+03 3.18E+03 3.90E+02 9.07E+02 4.24E+03 1.36E+03 181.975 0.05 Sb 8.63E+00 7.76E+00 6.01E+00 5.68E+00 5.10E+00 5.76E+00 6.30E+00 4.39E+00 5.04E+00 5.03E+00 8.67E+00 3.21E+00 4.60E+00 2.11E+01 206.836 0.0204 Se BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 8.39E+00 196.026 0.0375 Si 6.39E+02 4.43E+02 7.76E+02 5.06E+02 5.31E+02 3.35E+02 5.31E+02 3.39E+02 3.00E+02 1.00E+03 6.80E+02 4.20E+02 5.41E+02 BDL 5.04E+02 6.96E+02 Sn 2.00E+01 2.41E+01 2.11E+01 1.16E+01 1.48E+01 1.41E+01 1.24E+01 1.48E+01 1.47E+01 1.28E+01 2.16E+01 1.09E+01 2.23E+01 2.12E+01 189.927 0.0106 Sr 1.47E+02 1.27E+02 1.12E+02 8.17E+01 7.56E+01 9.66E+01 8.22E+01 9.06E+01 9.53E+01 7.72E+01 8.50E+01 9.53E+00 1.12E+02 1.10E+02 407.771 0.0042 Ti 4.83E+03 4.56E+03 4.10E+03 2.80E+03 3.01E+03 3.41E+03 3.09E+03 3.27E+03 3.18E+03 2.64E+03 2.75E+03 3.24E+02 4.04E+03 4.01E+03 334.94 0.0008 Tl BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 190.801 0.0367 V 2.04E+02 1.79E+02 1.70E+02 1.17E+02 1.24E+02 1.42E+02 1.27E+02 1.35E+02 1.34E+02 1.18E+02 1.42E+02 1.49E+01 1.50E+02 1.59E+02 290.88 0.0654 Zn 9.75E+02 1.55E+03 1.01E+03 7.81E+02 4.59E+02 3.71E+02 3.93E+02 4.67E+02 9.00E+02 4.83E+02 1.23E+03 1.87E+03 1.18E+03 6.77E+02 206.2 0.0011 BDL: below detection limit; nd: not determined.Continued 34 Analyte  3-12  3-16  3-20  3-24  3-28  4-0 4-16  5-0  5-4  5-8  5-12  5-16  5-20  5-24 5-28 MDL Ag BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 0.0024 Al 3.30E+04 3.34E+04 3.48E+04 3.33E+04 2.94E+04 1.52E+04 3.36E+04 8.16E+02 2.59E+04 2.84E+04 2.82E+04 3.06E+04 3.07E+04 2.93E+04 3.46E+04 0.0083 As BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 0.0361 Ba 4.44E+02 4.47E+02 4.59E+02 4.47E+02 2.55E+02 1.74E+02 4.34E+02 1.00E+01 2.20E+02 2.35E+02 2.11E+02 2.17E+02 1.98E+02 2.14E+02 4.44E+02 0.0006 Be 2.65E+00 2.74E+00 2.70E+00 2.46E+00 1.99E+00 1.24E+00 2.37E+00 BDL BDL BDL BDL BDL BDL BDL 2.42E+00 0.0005 Ca 1.08E+04 1.12E+04 1.10E+04 1.10E+04 1.70E+04 4.38E+03 7.82E+03 1.00E+03 7.54E+03 8.24E+03 8.26E+03 8.23E+03 7.74E+03 7.94E+03 9.51E+03 0.01 Cd BDL 1.71E+00 BDL BDL 4.66E+00 2.53E+00 1.39E+00 3.13E+00 1.35E+00 8.50E-01 6.15E-01 2.35E+00 3.30E+00 3.70E+00 1.40E+00 0.0019 Co 3.06E+01 3.22E+01 5.85E+02 2.93E+01 2.81E+01 1.47E+01 2.89E+01 1.00E+00 2.80E+01 3.13E+01 3.07E+01 3.27E+01 3.54E+01 3.75E+01 2.92E+01 0.0026 Cr 2.92E+01 3.19E+01 2.60E+01 2.47E+01 4.54E+01 1.98E+01 2.38E+01 4.81E+00 2.48E+01 2.71E+01 2.81E+01 2.73E+01 2.68E+01 2.72E+01 2.28E+01 0.0011 Cu 2.15E+02 1.77E+02 2.86E+02 1.13E+02 2.49E+02 1.43E+02 8.12E+01 6.24E+01 1.63E+02 1.55E+02 1.56E+02 1.09E+02 1.13E+02 1.29E+02 2.82E+02 0.0042 Fe 4.89E+04 4.79E+04 3.41E+04 2.89E+04 4.05E+04 3.50E+04 1.66E+04 9.13E+02 4.42E+04 4.53E+04 4.64E+04 4.82E+04 4.94E+04 4.73E+04 1.64E+04 0.0077 K 1.52E+04 1.51E+04 1.51E+04 1.41E+04 9.27E+03 6.08E+03 1.41E+04 3.17E+02 1.39E+03 1.39E+03 1.41E+03 1.58E+03 1.53E+03 1.43E+03 1.43E+04 0.05 Mg 2.28E+04 2.22E+04 2.22E+04 2.14E+04 2.00E+04 9.47E+03 2.09E+04 3.98E+02 1.85E+04 1.96E+04 2.03E+04 2.16E+04 2.13E+04 2.10E+04 2.10E+04 0.6521 Mn 1.16E+03 1.16E+03 1.15E+03 1.11E+03 9.52E+02 4.63E+02 1.09E+03 1.72E+01 9.75E+02 1.02E+03 1.04E+03 1.11E+03 9.79E+02 1.01E+03 1.10E+03 0.0136 Mo 1.67E+00 BDL 3.61E+00 BDL 2.25E+00 2.45E+00 BDL 1.44E+00 BDL BDL BDL BDL BDL BDL BDL 0.0058 Na 1.11E+03 9.13E+02 1.23E+03 1.28E+03 1.18E+03 5.14E+02 1.98E+03 BDL 1.27E+03 2.34E+03 1.37E+03 1.97E+03 1.92E+03 2.13E+03 1.59E+03 0.05 Ni 2.37E+01 3.89E+01 7.75E+02 2.19E+01 4.06E+01 1.63E+01 2.07E+01 3.56E+00 1.68E+01 1.68E+01 1.67E+01 1.83E+01 2.03E+01 1.96E+01 2.72E+01 0.004 S 5.49E+02 5.30E+02 7.44E+02 7.42E+02 3.87E+03 5.31E+03 9.75E+02 1.94E+03 na na na na na na 8.21E+02 0.05 Si 6.96E+02 6.04E+02 7.40E+02 3.40E+02 1.01E+03 1.33E+03 4.12E+02 5.02E+03 na na na na na na 7.89E+02 0.0029 P 1.07E+03 1.00E+03 7.78E+02 8.33E+02 2.61E+03 5.86E+02 8.76E+02 7.55E+01 na na na na na na 7.68E+02 0.05 Pb 2.15E+01 3.54E+01 2.21E+01 1.70E+01 3.51E+01 2.48E+01 4.65E+01 1.25E+01 8.76E+00 7.75E+00 7.76E+00 9.90E+00 1.13E+01 1.03E+01 1.29E+01 0.0204 Sb 6.89E+00 9.04E+00 5.98E+00 7.11E+00 1.11E+01 6.38E+00 7.45E+00 2.69E+00 BDL BDL BDL BDL BDL BDL 5.01E+00 0.0375 Se BDL BDL BDL BDL BDL BDL BDL 2.70E+00 BDL BDL BDL BDL BDL BDL BDL 6.96E+02 Sn 1.96E+01 2.71E+01 1.79E+01 2.23E+01 2.98E+01 1.62E+01 1.34E+01 8.26E+00 BDL BDL BDL BDL BDL BDL 1.50E+01 0.0106 Sr 9.65E+01 9.48E+01 9.57E+01 1.04E+02 1.06E+02 4.61E+01 8.95E+01 6.62E+00 8.02E+01 8.12E+01 8.11E+01 8.47E+01 8.15E+01 8.24E+01 9.90E+01 0.0042 Ti 3.88E+03 4.04E+03 3.96E+03 3.57E+03 2.76E+03 1.70E+03 3.42E+03 6.73E+01 2.85E+03 3.55E+03 3.72E+03 4.01E+03 3.62E+03 3.54E+03 3.48E+03 0.0008 Tl BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL 0.0367 V 1.46E+02 1.48E+02 1.48E+02 1.42E+02 1.29E+02 7.71E+01 1.36E+02 2.45E+00 1.20E+02 1.23E+02 1.25E+02 1.33E+02 1.04E+02 1.14E+02 1.37E+02 0.0654 Zn 9.60E+02 1.35E+03 7.86E+02 6.56E+02 2.16E+03 8.74E+02 4.01E+02 4.09E+02 2.15E+02 2.19E+02 2.16E+02 2.17E+02 3.46E+02 3.34E+02 7.60E+02 0.0011 BDL: below detection limit; na: not available  No Ag, As, Se, or Tl was detected. KC350a-org 0 ft4 ft8 ft12 ft16 ft20 ft24 ft28 ft36 ft24681 0D e p th  ( fe e t )Suspension pHKC350a-org 0 ft4 ft8 ft12 ft16 ft20 ft24 ft28 ft36 ft24681 0D e p th  ( fe e t )Leachate pHA B 36 KC350a-org 0 ft4 ft8 ft12 ft16 ft20 ft24 ft28 ft36 ft01 0 02 0 03 0 04 0 05 0 0D e p th  ( fe e t )Redox (mV)Figure 2.4 Chemical properties of KC samples from different depths compared to KC35 pH of clay suspensions (A); pH of prepared aqueous leachates of each sample (B); and redox potentials (C). The pH of both fresh aqueous suspensions and aqueous leachates of each sample were measured. The value for each individual sample as well as the mean have been plotted.    C Chapter 2: Characterization of Kisameet Clay  37   Biological properties      As the most abundant and diverse domain on the Earth, bacteria play crucial roles in different ecosystems in addition to their inseparable role in human health and disease (Fuks et al. 2018). They survive and even thrive, in a variety of environments and factor centrally in metabolic pathways that shape ecological, geological, and human health (Haruta and Kanno 2015; Svensson et al. 2017). Thus, characterizing resident bacterial populations is essential for providing a better understanding of any biological environments in terms of their ecology, chemistry, and homeostasis. KC is a very complex natural material, and many different factors may contribute to its unique properties, including biological properties such as its resident bacterial communities. Previously, Svensson et al. (2017) reported the detailed investigation of the bacterial communities that reside in the Kisameet Bay clay deposit by 16S rRNA metagenomic characterization. It was revealed that like soil, Kisameet clay samples harbor a complex mixture of bacterial taxa. Harboring an unexpected bacterial species diversity and richness, KC contains more than 300 species of bacteria in most samples, which increases to thousands of species for surface samples mixed with the organic overburden. As shown in Fig. 2.5, KC samples were dominated by Proteobacteria (88.3% of reads) (Svensson et al. 2017). Collectively, the KC deposit harbors several different bacterial communities, likely shaped by environmental parameters that change with depth.     Chapter 2: Characterization of Kisameet Clay 38 Figure 2.5 Bacterial diversity among KC samples Phylum- and class-level analysis of total assigned reads (identified by 16S rRNA metagenomics studies) for all KC samples. 88.3% of the reads belonged to Proteobacteria as the major bacterial phyla in KC samples. Further classification of Proteobacteria into constituent classes demonstrated that approximately 63.5% of reads belonged to Betaproteobacteria, followed by Gammaproteobacteria (21.3%), Alphaproteobacteria (4.0%), and Delta- proteobacteria (0.4%) (Svensson et al. 2017).  KC resident bacteria  2.3.4.1.1 KC resident bacteria identified by 16S rRNA sequencing         Using culture-based methods to study the cultivable portion of KC resident bacteria, we identified 117 bacterial strains isolated from a variety of KC samples (Table 2.4, and Table A.1). KC isolates were mostly identified as Proteobacteria (37.7%) followed by Firmicutes (33.3%), Actinobacteria (25.6%), and Bacteroidetes (3.4%). KC isolates include economically and medically important bacteria such as Actinomycetes, Pseudomonas, and Paenibacillus species.   Chapter 2: Characterization of Kisameet Clay 39 2.3.4.1.2 Phylogenetic tree of KC bacterial isolates      Most of our knowledge about bacterial diversity in different environments results from 16S rRNA gene sequencing as the most universally performed method for identification and phylogenetic studies (Bavykin et al. 2004).  Fig. 2.6 presents the phylogenetic tree of KC isolates. Antibacterial activity of KC samples among vertical cores     To gain insight into what geochemical properties might contribute to the antibacterial activity of KC, the inhibitory activity of KC core samples against E. coli MG1655 was compared to that of KC35. The activity of core samples showed high variability (Fig. 2.7). While KC35 exhibited strong antibacterial activity with approximately 5 log10 decrease in CFU within 24 h of incubation,  the core samples varied from bactericidal with greater than 3 log10 killing to more weakly active (show less than 1000-fold reduction in CFUs, but higher than water control alone). Some samples enhanced viability of the test organism compared to the water control. All samples from the interior cores (i.e., 4-12 ft) exhibited little or no activity. There was a very weak correlation between redox potential and antibacterial activity of KC samples (Table 2.2, Fig. 2.7), where samples with higher redox potential tend to show greater reduction of CFUs.  40 Table 2.4 117 KC isolates identified by 16S rRNA gene sequencing Kingdom Phylum Class Order Family Genus Bacteria Actinobacteria Actinobacteria Actinomycetales (25.6%) Dietziaceae Dietzia (1) Micrococcaceae Arthrobacter (14) Oerskovia (7) Microbacterium (3) Nocardiaceae Rhodococcus (3) Cellulomonadaceae Cellulomonas (1) Streptomycetaceae Streptomyces (1) Firmicutes Bacilli Bacillales (33.3%) Bacillaceae Bacillus (17) Exiguobacterium (2) Psychrobacillus (4) Planococcaceae Lysinibacillus (5) Paenibacillus (3) Sporosarcina (8) Proteobacteria Alphaproteobacteria Caulobacterales (1.7%) Caulobacteraceae Brevundimonas (2) Betaproteobacteria Burkholderiales (4.3%) Alcaligenaceae Achromobacter (2) Oxalobacteraceae Collimonas (1) Herminiimonas (1) Janthinobacterium (1) Gammaproteobacteria Pseudomonadales (21.4%) Moraxellaceae Enhydrobacter (1) Pseudomonadaceae Pseudomonas (24) Xanthomonadales (10.3%) Rhodanobacteraceae Rhodanobacter (5) Xanthomonadaceae Stenotrophomonas (7) Bacteroidetes Flavobacteria Flavobacterials (0.8%) Flavobacteriaceae Chryseobacterium (1) Sphingobacteria Sphingobacteriales (2.6%) Sphingobacteriaceae Sphingobacterium (3) Percentage of each order and the number of isolates belonging to each genus are presented in parentheses. Baldani et al. 2014; Mayilraj and Stackebrandi 2014. 41 Continued 42 Figure 2.6 Neighbour-joining tree based on the V2, V3, and V4 regions of 16S rRNA gene sequences, showing the phylogenetic relationship among the KC isolates The phylogenetic tree of KC isolates was assembled by the neighbour joining method in MacVector (v.13.0) using 16S rRNA gene sequence of Escherichia coli K12 as a reference. Genetic distances were generated using the Kimura 2-parameter method. Tie breaking equals Random Distance: Uncorrected ("p") Gaps distributed proportionally.Chapter 2: Characterization of Kisameet Clay 43 KC350a-org0 ft4 ft8 ft12 ft16 ft20 ft24 ft28 ft36 ft1 0 -11 0 01 0 11 0 21 0 31 0 41 0 51 0 6D e p th  ( fe e t )Activity againstE. coli MG1655(Fold reduction in CFUs)1000 foldKC35dH2OBufferKis2Kis3Kis4Kis5Kis1KC35Figure 2.7 Antibacterial activity of suspensions of KC core samples against E. coli MG1655 Results show the mean fold-reduction in CFUs compared to the inoculum for three independent trials. The mean fold reduction for each sample (Kis1 to Kis5) at different depth has been compared to the antibacterial activity of KC35 (a bucket sample) as a positive control and dH2O as a negative control. Error bars represent the standard error (SE) of the mean of three independent replicates of each sample. Stronger antibacterial activity was detected at the edges of the deposit. Discussion     Characterization of KC samples obtained from different locations and depths revealed heterogeneity across the Kisameet Bay deposit in terms of the physicochemical properties. Our results also revealed variation of the antibacterial properties of KC across the deposit. This natural heterogeneity has been reported previously for the OMT clay deposit (Morrison et al. 2016). The elemental analyses of bulk clay samples confirmed the previous study, identifying KC as an Fe and Al rich clay mineral (Hauser 1951). Chapter 2: Characterization of Kisameet Clay 44 Moreover, while all the natural clay minerals with potent antibacterial activity were reported to contain smectite as the dominant mineral group (Williams et al. 2009; Morrison and Williams 2014, Londono and Williams 2015; Otto and Haydel 2013a), KC contains biotite as the major clay mineral. Morrison and Williams (2014) reported that the mineralogy of the OMT deposit was dominated by mixed-layered illite-smectite clay minerals (43-53%) followed by quartz (36-49%). KC also contains quartz (6.8-22.5%) as the major non-clay mineral. Additionally, we could not detect any pyrite (a reduced iron-phase) in KC samples as reported for other antibacterial clays (Morrison and Williams 2014; Londono and Williams 2015; Otto and Haydel 2013a).       Svensson et al. reported that KC is a rich source of economically valuable bacterial species including Actinobacteria. A large number of soil Actinobacteria accounts for 70-80% of commercially available secondary metabolites and more than 50% of the known antibiotics identified to date (Kumar and Jadeja 2016). While up to 3% of reads in the KC microbiome analysis were assigned to the class of Actinobacteria, 25% of the KC isolates, obtained by culture-based methods, belonged to the order of Actinobacteria that included Arthrobacter and Streptomyces species, both well-known producers of antibiotics and antimicrobial secondary metabolites (Kamigiri et al. 1996). Moreover, 20.5% of KC isolates were identified as members of the family Pseudomonadaceae with remarkable physiologic and metabolic versatility and broad potential for adaptation to fluctuating environmental conditions, which together facilitate their colonization in diverse niches (Silby et al. 2011). Although natural products have been an inexhaustible source of novel therapeutic agents and bioactive compounds (Davies 2010), they require rigorous scientific analysis to support their efficacy and to define their complex nature and active principal components. As natural products have played a vital role in antibiotic discovery and development (Newman and Cragg 2016), further investigation of the KC isolates which may have interesting potential as producers of novel bioactive compounds or as biocontrol/bioremediation agents is necessary.45  Antimicrobial Properties of Kisameet Clay Introduction            Natural clay minerals with a long history of therapeutic applications have recently received significant interest for their beneficial properties in combatting infectious diseases (Williams et al. 2004; Haydel et al. 2008). Among therapeutic clay minerals, only a few clays have been recognized as antibacterial with potent killing effect against a broad-spectrum of human pathogens (Morrison et al. 2016). Ure et al. (1946) reported the inhibitory action of KC suspensions (Ray-Vite, 25% wt/vol) on the growth of E. coli and S. aureus in liquid cultures;  while  a large dose of KC suspensions inhibited the growth of both bacteria, application of small doses caused slow and poor growth. Our preliminary experiments using 1% aqueous suspensions of KC revealed the antibacterial activity of some samples (Fig. 2.7). The goal of this chapter is to investigate the antimicrobial activity of KC against a variety of bacterial and fungal pathogens and to characterize the spectrum of activity. In addition, a variety of MDR clinical isolates was tested to see how development of resistance mechanisms might affect the sensitivity of bacteria to KC preparations.  Materials and methods Microbial strains and growth conditions      The bacterial and fungal strains used in this study are described in Tables 3.1 and 3.2. E. coli MG1655, S. aureus RN4220, and P. aeruginosa PAO1 were used as representative Gram-negative, Gram-positive,and clinically important organisms, respectively; while Mycobacterium marinum M strain was investigated as the most closely related Mycobacterial species to M. ulcerans, the causative agent of Buruli ulcer (Stinear et al. 2000; Doig et al. 2012; Chany et al. 2013). E. coli MG1655 and P. aeruginosa PAO1 were grown in LB broth or on LB agar, while S. aureus RN4220 was grown in tryptic soy broth (TSB) or on tryptic soy agar (TSA). M. marinum was grown in 7H9 broth or 7H10 agar media (Difco) Chapter 3: Antimicrobial Properties of Kisameet Clay  46  supplemented with 1% OADC (oleic acid, albumin, dextrose, and catalase) (Middlebrook), and 0.2% glycerol.       To study the activity of KC against MDR bacterial pathogens, the main causative agents of nosocomial infections worldwide, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) were investigated (Rice 2008). A collection of 16 strains from a number of sources in Vancouver, including Vancouver General Hospital (VGH), St. Paul’s Hospital (SPH), and the University of British Columbia (UBC) wastewater treatment pilot plant (WWTP) were studied (Table 3.1). In addition, to investigate whether KC may have value for the treatment of a range of serious bacterial pathogens involved in chronic pulmonary infections in cystic fibrosis (CF) patients, a further study was conducted on 17 clinical isolates consisting of twelve B. cepacia complex (BCC), four P. aeruginosa, and one Stenotrophomonas maltophilia, isolated from cystic fibrosis patients and deposited at the Canadian BCC Research and Referral Repository at UBC, Vancouver, Canada. All these isolates were collected from CF patients attending the adult or pediatric CF clinics in Vancouver, British Columbia between 1990-2015 (Table 3.2). E. faecium strains were grown in Mueller-Hinton (MH) broth or on MH agar, while S. aureus and MRSA USA300 were grown in TSB or on TSA plates. All other ESKAPE strains as well as CF isolates (Table 3.2) were cultured in LB broth or on LB agar. All bacterial liquid cultures were incubated at 37˚C with gentle orbital rotary shaking. For antifungal studies, two pathogenic fungi, C. albicans SC5314, ATCC MYA-2876 and C. neoformans H99 were studied. C. albicans was grown in TSB or on TSA plates and C. neoformans in YPD (1% yeast extract, 2% peptone, 2% dextrose) (wt/vol) broth or on YPD agar at 30˚C and 37˚C.    47 Table 3.1 Bacterial and fungal strains used in this study Accession numbers indicate strains identified by 16S rRNA gene sequencing. No. Species Strain Source Media Temp References 1 Escherichia coli K12 MG1655 JED collection LBB, LBA 37˚C Bachmann 19722 Staphylococcus  aureus RN4220 JED collection TSB, TSA 37˚C Kreiswirth et al. 19833 Pseudomonas aeruginosa PAO1 JED collection LBB, LBA 37˚C Holloway 19694 Mycobacterium marinum JVC1704, (M strain) JED collection 7H9, 7H10 + OADC 30˚C Ramakrishnan et al. 19945 Enterococcus faecium BM4145 JED collection MHB, MHA 37˚C Rende-Fournier et al. 19936 E. faecium NCBI-P15 WWTP MHB, MHA 37˚C Accession no. KT8274007 S. aureus MRSA USA300 JED collection TSB, TSA 37˚C Tenover and Goering  20098 S. aureus SA-8325-4 JED collection TSB, TSA 37˚C Lin and Davies 20079 Klebsiella pneumoniae KP-1247 VGH LBB, LBA 37˚C 10 K. pneumoniae KP-1771 SPH LBB, LBA 37˚C 11 K. pneumoniae KP-1772 SPH LBB, LBA 37˚C 12 K. pneumoniae KP-1777 SPH LBB, LBA 37˚C 13 K. pneumoniae KP-1780 SPH LBB, LBA 37˚C 14 Acinetobacter baumannii AB-1264  VGH LBB, LBA 37˚C 15 A. baumannii AB-1270  VGH LBB, LBA 37˚C 16 P. aeruginosa PA-1245 SPH LBB, LBA 37˚C 17 P. aeruginosa PA-1251 VGH LBB, LBA 37˚C 18 Enterobacter sp. NCBI-MI1 WWTP LBB, LBA 37˚C Accession no. KT82739819 Enterobacter sp. NCBI-MI16 WWTP LBB, LBA 37˚C Accession no. KT827399 20 Enterobacter sp. E. cloacae-1172 JED collection LBB, LBA 37˚C Xu et al. 201121 Candida albicans SC5314, ATCC MYA-2876  JED collection TSB, TSA 30˚C, 37˚C Gillum et al. 198422 Cryptococcus neoformans H99 Kronstad Lab, UBC YPD broth, YPD agar 30˚C, 37˚C Franzot et al. 199948 Table 3.2 Clinical pathogens isolated from cystic fibrosis patients a, b, c  sequential isolates from the same patients that were previously evaluated for strain type by random amplified polymorphic DNA (RAPD) analysis (Speert et al. 2002b) d two morphotypes of the strainVC15184: (-1, mucoid and -2, non- mucoid strain which was recovered during the course of this study and confirmed to have the same RAPD phenotype).   The remaining strains were independent isolates from one patient. e Resp indicates a sample from respiratory tract, either sputum or throat/cough swab. No.  Species Strain Year Source Temp References 1 Burkholderia cepacia VC9490 1999 Sputum 37˚C 2 Burkholderia cenocepacia C3921a  1990 Sputum 37˚C Zlosnik and Speert 2010; Miller et al. 20153 Burkholderia cenocepacia C8963a2000 Respe 37˚C Zlosnik and Speert 2010; Miller et al. 20154 Burkholderia cenocepacia C9343a 2000 Resp 37˚C Zlosnik and Speert 2010; Miller et al. 20155 Burkholderia cenocepacia VC13195b 2006 Resp 37˚C 6 Burkholderia cenocepacia VC15185b 2010 Resp 37˚C 7 Burkholderia cenocepacia VC15442b 2010 Blood 37˚C 8 Burkholderia dolosa VC14902 2009 Sputum 37˚C 9 Burkholderia multivorans VC5602c 1993 Sputum 37˚C Silva et al. 2016   10 Burkholderia multivorans VC16929c 2013 Sputum 37˚C Silva et al. 201611 Burkholderia stabilis VC7909 1993 Sputum 37˚C 12 Burkholderia vietnamiensis VC9237 1998 Resp 37˚C Kennedy et al. 201613 Pseudomonas aeruginosa VC8263, A002 type 1997 Resp 37˚C Speert et al. 2002a14 Pseudomonas aeruginosa VC15184-1d  2010 Resp 37˚C 15 Pseudomonas aeruginosa VC15184-2d  2010 Resp 37˚C 16 Pseudomonas aeruginosa VC17829, A097 type 2015 Sputum 37˚C Speert et al. 2002a17 Stenotrophomonas maltophilia VC13512 2006 Sputum 37˚C Chapter 3: Antimicrobial Properties of Kisameet Clay 49 Kisameet clay samples       The unprocessed natural clay mineral in its original wet form was dried in a vacuum desiccator at room temperature or by heating in an oven at 60˚C. Dry KC samples were ground by mortar and pestle and autoclaved at 121˚C for 1 h before testing. Measurement of pH was performed using equilibrated suspensions of 1g clay in 10 mL dH2O or aqueous leachates with a VWR-SB20 pH meter.   KC suspensions and aqueous leachates preparations      Aqueous suspensions of KC with 1, 5, or 10% (wt/vol) concentrations were prepared by suspending 10, 50, or 100 mg of dry, ground, autoclaved clay in 1 mL of sterile dH2O, respectively. KC aqueous leachates (L50, L100, and L500) were obtained by adding 1, 2, or 10 g of autoclaved KC to 20 mL of sterile dH2O resulting in 5, 10, 50% (wt/vol) aqueous suspensions, respectively. After continuous agitation for 24 h at room temperature, suspensions were then centrifuged at 25,000 rpm for 2 h at 4˚C to separate insoluble minerals from the leachate solution. The supernatants were clarified and sterilized by passage through a 0.22 μm filter (Millipore).  Elemental analysis of KC aqueous leachates by ICP-OES      Measurement of the elemental concentration of the KC leachates was determined by ICP-OES using a Perkin Elmer Optima 7300 DV spectrometer equipped with a Scott spray chamber and Gem Tip Cross-Flow nebulizer. Calibration standards were prepared from certified multi-element stock solutions (Multi Element Calibration Standard 3, Perkin Elmer Inc., 4400-010 Quality Control Standard-21 elements, Atomic Spectroscopy Standard) except for Si, S, and P, which were single element standards. A working calibration curve of at least seven measurements was prepared by diluting stock solutions in 0.5% nitric acid. Three KC leachate samples, blank (dH2O used for preparing leachates), and standards were also diluted with 0.5% nitric acid solution until their response was determined to be within the calibration range. Internal standards (Y) were added to both standards and samples prior to analysis.   Chapter 3: Antimicrobial Properties of Kisameet Clay  50   Antibacterial agents and susceptibility assays      The antibacterial resistance profiles of clinical isolates were characterized by standard agar disk diffusion susceptibility assays (Bauer et al. 1966), according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2011). Susceptibility assays were performed using cation-adjusted MH-II broth and agar media and a panel of 40 antibiotics representing antibacterial agents from more than 16 different classes (Table 3.3). Briefly, an overnight culture of each isolate in MH broth was diluted and incubated with gentle shaking to reach mid-exponential phase of growth. MH agar plates were then inoculated with the bacterial cultures and antibiotic disks (Oxoid, BBL) were placed on the inoculated plates and incubated at 37˚C for 20-24 hours, before the zones of inhibition (ZOI) were measured.           51 Table 3.3 Antibacterial agents used in this study Class Antibiotics Aminoglycosides Amikacin (10)b; Gentamicin (10); Kanamycin (30); Neomycin (30); Streptomycin (10); Tobramycin (100) Aminocyclitols Spectinomycin (100)b Carbapenems Ertapenem (10); Imipenem (10); Meropenem (10) 1st generation cephalosporins Cephalothin (30); Cephazolin (30) 2nd generation cephalosporins Cefuroxime (30); Cefotetan (30); Cefoxitin (30) 3rd generation cephalosporins Ceftazidime (30); Cefixime (5); Cefpodoxime (10); Ceftriaxone (30); Cefotaxime (30) Glycopeptides Vancomycin (30)a Lincosamides Clindamycin (2)a Macrolides Erythromycin (15)a Nitrofurans Nitrofurantoin (300) Penicillins Amoxicillin-clavulanic acid (30); Ampicillin (10); Methicillin (5)a; Oxacillin (1)a; Piperacillin (100)b Polypeptides Colistin (10); Polymyxin B (300) Phenicols Chloramphenicol (30) Quinolones Ciprofloxacin (50); levofloxacin (5); Nalidixic acid (30) Sulfonamides Sulfadiazine (250)b; Sulfamethoxazole-trimethoprim (25) Tetracyclines Doxytetracycline (30); Tetracycline (30) Trimethoprim Trimethoprim (5) Amount (µg) per disk of antibiotics (Oxoid, BBL) is indicated in parentheses. a Antibiotic used only for Gram-positive members of ESKAPE isolates; b antibiotic used only for CF isolates. Chapter 3: Antimicrobial Properties of Kisameet Clay  52   Antimicrobial assays of KC aqueous suspensions and leachates  Viability assays      An in vitro assay was used to investigate the effect of KC on microbial strains. Briefly, aqueous suspensions of KC with 1, 5 or 10% (wt/vol) concentrations were prepared by suspending 10, 50, or 100 mg of dry, ground, and autoclaved clay in 1 mL of sterile dH2O, respectively. Overnight cultures of bacterial or yeast strains were diluted into the fresh appropriate growth medium to an approximate concentration of ~107 CFU mL-1, based on OD600, and incubated at 37˚C (bacteria and fungi) or 30˚C (fungi) with gentle rotary mixing at 200 rpm to reach mid-exponential phase of growth based on growth curves. Cells were harvested by centrifugation, rinsed once in sterile phosphate-buffered saline (PBS) (pH 7.4) and resuspended in either a suspension of KC in dH2O (1 or 10% (wt/vol) for bacterial strains and 5% (wt/vol) for fungal strains), or in dH2O only (to study the viability of organisms in the absence of KC) at an initial cell concentration of ~107 CFU mL-1. Suspensions were then incubated at the appropriate temperature as described above with shaking at 200 rpm to prevent sedimentation and to provide contact with the clay minerals or aqueous leachates. Antibacterial assays with different leachates were performed in the same way. For leachate experiments, phosphate buffers with a low-pH, comparable to that of KC leachates, were used as negative controls. Viability was determined by 10-fold serial dilution plating of aliquots removed at the start of experiments for initial CFUs and at different time points following exposure to clay suspensions (5 or 24 h of incubation for bacterial cells except CF isolates and 24, and 48 h for CF isolates and fungal strains). For leachate experiments, CFUs were determined at appropriate time points within 24 h of exposure.   Growth inhibition assays for M. marinum      Mid-exponential phase growth of M. marinum in 7H9 broth supplemented with 1% OADC and 0.2% glycerol was treated with 250 mg mL-1 (25% wt/vol) KC and incubated at 30˚C. The inhibitory activity was examined during 12 days by determining the viability of bacteria treated with KC compared to the Chapter 3: Antimicrobial Properties of Kisameet Clay  53  growth of bacteria in the same media without treatment as a control. Viability was determined by 10-fold serial dilution plating of aliquots removed at the start of experiments for initial CFUs on 7H10 agar plates following 0, 2, 5, 12 days of exposure to clay. In addition, the inhibitory action of KC was studied by supplementing 7H10 agar medium with 25% (wt/vol) dry KC.   Organic solvent extracts of KC      Ten grams of original wet KC were shaken with 30 mL ethanol, ethyl acetate, or hexane for 24 h at room temperature. Liquid phases were separated from mineral particles, evaporated, and then re-suspended in 200 µL dimethyl sulfoxide (DMSO). Sterile paper disks (6 mm) were impregnated with 20 µL of each extract and dried. The antibacterial activity of extracts was investigated in standard disk diffusion assays using mid-exponential phase growth of E. coli MG1655, S. aureus RN4220, P. aeruginosa PAO1, and MRSA on TS agar plates.     Results  Antibacterial activity of KC aqueous suspensions       To determine the spectrum of KC activity and provide insight into its possible mode of action, the inhibitory effect of KC against a group of representative Gram-negative, Gram-positive, and clinically important bacterial strains, E. coli, S. aureus, and P. aeruginosa, respectively, was investigated using in vitro antimicrobial susceptibility assays. KC exhibited broad-spectrum antibacterial activity against different bacteria (Fig. 3.1). Incubation of E. coli MG1655 with a 1% aqueous suspension of KC (Fig. 3.1A) resulted in an approximately 3 log10 reduction in CFUs after 5 h, while 24 h of treatment completely eliminated the number of viable bacteria to below the limit of detection. In comparison, no significant decrease in survival was observed for the bacteria incubated in water alone. Similarly, the Gram-positive S. aureus and the antimicrobial tolerant P. aeruginosa were completely eliminated within Chapter 3: Antimicrobial Properties of Kisameet Clay  54  24 h of incubation (Fig. 3.1B, C). As for E. coli, in the first 5 h of exposure, P. aeruginosa viability was reduced by ~3.5 log10, while S. aureus showed ~1.5 log10 reduction in CFUs compared to the control.     E . c o li  M G 1 6 5 5Log10 CFU/mL0 524 0 524012345678T im e  (h )^S . a u re u s  R N 4 2 2 0Log10 CFU/mL0 524 0 524012345678T im e  (h )^P . a e ru g in o s a  P A O 1Log10 CFU/mL0 524 0 524012345678dH2O1% KCT im e  (h )^ Figure 3.1 Viability of E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) after treatment with 1%  (wt/vol) aqueous suspensions of KC CFUs were determined after 0, 5, and 24 h of incubation. The dotted line at log10=1 of the Y axis represents the limit of detection for CFU.  ^ indicates that viable cells were below the limit of detection at that time point.  Error bars represent the standard error (SE) of the mean of at least three independent replicates.         Antibacterial activity of KC aqueous leachates      Previously, exchangeable metal ions have been reported to be responsible for the activity of some antibacterial clays (Otto and Haydel 2013a; Morrison et al. 2016). To identify the chemical components underlying the broad-spectrum antibacterial activity of KC and to investigate whether the exchangeable/soluble fraction of KC is involved in the inhibitory activity, aqueous leachates (L50, L100, and L500, made from 50, 100, and 500 mg mL-1 KC suspensions in water) were prepared as described in C A B Chapter 3: Antimicrobial Properties of Kisameet Clay  55  section 3.2.3. As KC leachates were acidic (pH 3.5-3.8, depending on the concentration of clay suspensions used in the preparation of leachates) (Table 3.4), the contribution of the low-pH stress of KC alone on the viability of the bacterial strains was also tested by comparing the antibacterial activity of the three KC leachates with phosphate buffers of comparable pH (3.5 and 3.8).        Antibacterial assays using KC leachates against E. coli MG1655, S. aureus RN4220, and P. aeruginosa PAO1 revealed strong bactericidal activity, which increased with the amount of clay used to make the leachate. Fig. 3.2A shows that L500 and L100 completely eliminated E. coli after 1 h of incubation, while L50 showed complete bactericidal activity within 4 h. In comparison, both low-pH buffers caused less than a 1 log10 decrease in the viability of E. coli in the first 4 h, suggesting that leachate activity was not solely due to the low pH. Similarly, as shown in Fig. 3.2C, L500 showed complete killing against P. aeruginosa in 1 h, while L100 and L50 showed full bactericidal activity within 4 h of incubation. Although P. aeruginosa exhibited higher sensitivity to low-pH buffers compared to E. coli, (Fig. 3.2A, C), its viability was also reduced to 1-2.5 log10 within first 4 h of exposure. The viability of S. aureus treated with L500 was reduced 5 log10 in the first 5 h and was completely eliminated within 8 h of incubation (Fig. 3.2B). Moreover, no viable bacteria were recovered from S. aureus suspensions treated with L100 and L50 after 24 h. However, while S. aureus showed lower sensitivity to low-pH buffer (pH 3.5) compared to L500, the buffer with pH 3.8 decreased the number of recovered viable S. aureus more than L100 and L50 after 8 h of incubation. Together, this shows that a component responsible for the activity of KC against a variety of bacteria is water soluble, and that while low pH is a characteristic of KC leachates, it does not appear to be solely responsible for their activity, under the conditions studied.        Elemental analysis of KC leachates by ICP-OES (Table 3.4) provided more insight into the composition of leachates and revealed the mM concentrations of elements such as Al, Ca, Fe, Mg, Na, S, 56 and Si in KC leachates while Ag, B, Cd, Mo, P, Pb, Sb, Se, Ti, Tl, and V were all below the detection limits.  E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mL0 4 8 1 2 1 6 2 0 2 4012345678dH2OL500L100L50Buffer ( 3.5)Buffer ( 3.8)S . a u re u s  R N 4 2 2 0T im e  (h )Log10 CFU/mL0 4 8 1 2 1 6 2 0 2 4012345678dH2OL500L100L50Buffer ( 3.5)Buffer ( 3.8)A B Chapter 3: Antimicrobial Properties of Kisameet Clay 57 P . a e ru g in o s a  P A O 1T im e  (h )Log10 CFU/mL0 4 8 1 2 1 6 2 0 2 4012345678dH2OL500L100L50Buffer ( 3.5)Buffer ( 3.8)Figure 3.2 Antibacterial activity of three KC aqueous leachates (L50, L100, and L500) against E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) compared to low-pH phosphate buffers The dotted line at log10=1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates.     Antibacterial activity of KC organic extracts     In addition to the antibacterial activity of aqueous leachates, KC was also tested for the presence of active organic compounds, which may also contribute to its activity. Organic extracts of KC were prepared using solvents of different hydrophobicity (ethyl acetate, ethanol, and hexane) and tested for their activity in a standard susceptibility disk diffusion assay. As shown in Table 3.5, KC extracts inhibited two Gram-positive and drug-resistant bacterial strains, S. aureus RN4220 and MRSA USA300, but not E. coli MG1655 or P. aeruginosa PAO1. Further chemical investigation of the ethyl acetate extracts of dry KC were then fractionated using thin-layer chromatography (TLC) (Tang J, Davies J, unpublished data). Further chemical investigation identified the presence of elemental/cyclo sulfur-8 (S8) which has been shown previously to be antibacterial (Weld and Gunther 1947; Libenson et al. 1953).  C 58 Table 3.4 Elemental analysis of KC aqueous leachates (L50, L100, and L500) by ICP-OES Element   L50  L100  L500 MDL Analyte µg/L µM µg/L µM µg/L µM     µg/L (emission nm) Ag BDL  - BDL  - BDL  - 2.4 Ag 328.068 Al 5.54E+03 205.27 1.39E+04 514.73 2.82E+04 1,045.56 8.3 Al 396.153 As 1.46E+01 0.20 3.00E+00 0.04 1.40E+00 0.02 36.1 As 188.979 B BDL - BDL - BDL - 1.0 B 249.677 Ba 6.69E+00 0.05 6.61E+00 0.05 5.90E+00 0.04 0.6 Ba 233.527 Be 5.42E+00 0.60 9.72E+00 1.08 1.63E+01 1.81 0.5 Be 313.107 Ca 7.77E+04 1938.24 1.15E+05 2865.95 1.18E+05 2,933.05 10.0 Ca 317.933 Cd BDL - BDL - BDL - 1.9 Cd 228.802 Co 9.22E+01 1.56 1.30E+02 2.20 2.15E+02 3.65 2.6 Co 228.616 Cr 4.55E+00 0.09 2.13E+00 0.04 1.23E+00 0.02 1.1 Cr 267.716 Cu 1.04E+02 1.63 1.54E+02 2.43 2.09E+02 3.29 4.2 Cu 327.393 Fe 2.42E+04 433.70 2.87E+04 513.59 5.08E+04 908.85 7.7 Fe 238.204 K 2.81E+03 71.84 3.15E+03 80.66 4.02E+03 102.94 50.0 K 766.490 Li 8.79E+01 12.66 1.80E+02 25.98 2.99E+02 43.12 0.3 Li 670.784 Mg 4.36E+04 1792.93 6.26E+04 2577.12 7.43E+04 3,056.71 652.1 Mg 285.213 Mn 2.79E+03 50.84 4.19E+03 76.24 6.62E+03 120.41 13.6 Mn 257.610 Mo BDL - BDL - BDL - 5.8 Mo 202.031 Na 9.28E+03 403.85 1.29E+04 559.85 2.10E+04 911.81 50.0 Na 589.592 Ni 5.14E+01 0.88 8.73E+01 1.49 1.13E+02 1.93 4.0 Ni 231.604 P BDL - BDL - BDL - 50.0 P 213.617 Pb BDL - BDL - BDL - 2.9 Pb 220.353 S 3.59E+04 1119.92 1.01E+05 3162.65 3.02E+05 9,406.18 50.0 S 181.975 Sb BDL - BDL - BDL - 20.4 Sb 206.836 Se BDL - BDL - BDL - 37.5 Se 196.026 Si 5.62E+03 200.23 8.38E+03 298.39 9.34E+03 332.54 10.0 Si 251.611 Sn 8.70E+00 0.07 1.08E+01 0.09 2.82E+02 2.38 10.6 Sn 189.927 Sr 2.71E+02 3.10 3.96E+02 4.52 4.54E+02 5.18 4.2 Sr 407.771 Ti BDL - BDL - BDL - 0.8 Ti 334.940 Tl BDL - BDL - BDL - 36.7 Tl 190.801 V BDL - BDL - BDL - 65.4 V 290.880 Zn 9.53E+02 14.58 4.60E+02 7.03 5.72E+02 8.75 1.1 Zn 206.200 pH       3.77-3.82      3.60-3.64       3.47-3.51 Aqueous leachates of KC, L50, L100, and L500, were prepared from different concentrations of aqueous suspensions of KC (50, 100, and 500 mg mL-1 respectively). dH2O used in leachates preparation was applied as a blank and the amounts were normalized with blank. Ag, B, Cd, Mo, P, Pb, Sb, Se, Ti, Tl, and V were all below the detection limits for three samples of leachates. MDL: Minimal detection limit; BDL: Below detection limit   Chapter 3: Antimicrobial Properties of Kisameet Clay 59 Table 3.5 Antibacterial activities of KC organic extracts against four bacterial strains Bacteria Hexane Ethyl acetate Ethanol S. aureus RN4220 12 11 10 MRSA USA300 12 11 10 E. coli MG1655 0 0 0 P. aeruginosa PAO1 0 0 0 Disk diffusion assays using 6-mm paper disks impregnated with organic solvent extracts of KC against four bacterial strains; ZOI was measured from the edge of the disks and recorded in mm.  Antibacterial activity of KC against clinical MDR bacterial isolates ESKAPE pathogens 3.3.4.1.1 ESKAPE pathogens and their clinical importance      While the discovery of potent antimicrobial agents has arguably led to significant health care advances, the genetic flexibility of major human bacterial pathogens has compromised the effectiveness of these therapeutics (Rice 2008; Lewis 2017). A coterie of certain pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has been shown worldwide to cause the majority of nosocomial infections, and to effectively “escape” the lethal action of available antibacterial agents (Rice 2008). Rice collectively referred this group as “the ESKAPE bugs” as they are responsible for the majority of nosocomial infections and present paradigms of increasing prevalence (within nosocomial settings), transmission, pathogenesis and resistance. They are so named since they “escape” the activity of all available antimicrobial agents and cause extensive morbidity and mortality in infected patients (Rice 2008; Boucher et al. 2009). They are predicted to be of increasing relevance in infectious disease for the foreseeable future, but the current antibiotic armamentarium has little to offer in terms of treatment, and there are few novel antimicrobial agents under development that show promise in relieving the health Chapter 3: Antimicrobial Properties of Kisameet Clay 60 crisis caused by these organisms (Pendleton et al. 2013). Following studies of the antibacterial activity of KC against bacterial lab strains, to investigate its activity against MDR bacterial pathogens, which developed a variety of resistance mechanisms, the ESKAPE strains were tested.  3.3.4.1.2 Antibiotic resistance patterns of ESKAPE pathogens      Standard disk diffusion tests using a panel of 36 antibiotics showed widespread, although variable, multidrug resistance among these strains (Table 3.6). The E. faecium and S. aureus strains exhibited resistance to carbapenems, third-generation cephalosporins, and penicillins, while MRSA USA300 was also resistant to first-generation cephalosporins, quinolones, tetracyclines, nitrofurantoin, clindamycin, and erythromycin. All Gram-negative strains were resistant to first- and second-generation cephalosporins and penicillins. In addition, K. pneumoniae, A. baumannii, and P. aeruginosa strains exhibited resistance to third-generation cephalosporins and trimethoprim (Behroozian et al. 2016). 3.3.4.1.3 Antibacterial activity of KC suspensions against ESKAPE pathogens      Using an in vitro assay to study the effect of KC on the ESKAPE strains revealed that KC dramatically reduced the viability of all strains tested (Fig. 3.3).  For example, after 5 h exposure to KC, no viable cells of A. baumannii AB-1270, Enterobacter MI1, or Enterobacter MI16 could be recovered, indicating potent activity against these strains. In addition, S. aureus and K. pneumoniae, P. aeruginosa, A. baumannii AB-1264, and E. cloacae-1172 lost viability completely after 24 h, and the same killingtook 48 h for E. faecium strains. In contrast, in water-only controls without KC, the decline in CFU during the same period of incubation was ≤ 1 log10 for all Gram-negative strains and ~1-3 log10 for E. faecium and S. aureus strains, respectively (Behroozian et al. 2016). 61 Table 3.6 Resistance patterns of ESKAPE strains for different classes of antibiotics Filled black circles indicate resistance (zone of inhibition ≤ 1 mm from the edge of disk of antibiotics); no mark indicates wider zone of inhibition. Gray boxes indicate no test was conducted. Amount (µg) per disk of antibiotics (Oxoid, BBL) is indicated in parentheses.   Colistin and polymyxin B –polypeptides- active against only Gram-negative bacteria were also tested on K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp.; no resistance was observed (Behroozian et al. 2016).Species Strain Gentamicin (10)Kanamycin (30)Neomycin (30)Streptomycin (10)Tobramycin (10)Ertapenem (10)Imipenem (10)Meropenem (10)Cephalothin (30)Cephazolin (30)Cefuroxime (30)Cefotetan (30)Cefoxitin (30)Ceftazidime (30)Cefixime (5)Cefpodoxime (10)Ceftriaxone (30)Cefotaxime (30)Amoxicillin-clavulanic acid (30)Ampicillin (10)Methicillin (5)Oxacillin (1)Ciprofloxacin (5) Levofloxacin (5) Nalidixic acid (30)Sulfamethoxazole-  trimethoprim (25)Trimethoprim (5)Doxycycline (30)Tetracycline (30)Chloramphenicol (30)Nitrofurantoin (300)Vancomycin (30)Clindamycin  (2)Erythromycin (15) SulfonamideE. faecium BM4145 • • • • • • • • • • • • • • • • • • • •NCBI-P15 • • • • • • • • • • •S. aureus MRSA USA300 • • • • • • • • • • • •SA-8325-4 • • • • •K. pneumoniae KP-1247 • • • • • • • • • • • • • • • • • •Aminoglycosides Carbapenems 1st, 2nd, 3rd generation cephalosporins Penicillins Quinolones TetracyclinesKP-1771 • • • • • • • • • • • • • • • •KP-1772 • • • • • • • • • • • • • • • • • • •KP-1777 • • • • • • • • • • • • • • • • • • •KP-1780 • • • • • • • • • • • • • • •A. baumannii AB-1264  • • • • • • • • • • • • •AB-1270  • • • • • • • • • • • • •PA-1245 • • • • • • • • • • • • • • • • • • • • • • • • •PA-1251 • • • • • • • • • • • • • • • • • • • • • • • • • •Enterobacter spp. NCBI-MI1 • • • • • • •NCBI-MI16 • • • • • • • •E. cloacae -1172 • • • • • • • •P. aeruginosa62 E . fa e c iu mT im e  (h )Log10 CFU/mL0 52448 0 52448012345678B M 4145E F -P 1 5^ ^S . a u r e u sT im e  (h )Log10 CFU/mL0 524 0 524012345678M R S A  U S A 300S A -8 3 2 5 -4^ ^K . p n e u m o n ia eT im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524012345678 K P -1 2 4 7K P -1 7 7 1K P -1 7 7 2K P -1 7 7 7K P -1 7 8 0^ ^^ ^ ^A B C 63 A . b a u m a n n i iT im e  (h )Log10 CFU/mL0 524 0 524012345678A B -1 2 64A B -1 2 70^ ^ ^P . a e r u g in o s aT im e  (h )Log10 CFU/mL0 524 0 524012345678P A -1 2 45P A -1 2 51^ ^E n te r o b a c te r  s p p .T im e  (h )Log10 CFU/mL0 524 0 524 0 524012345678MI1MI16E . c lo a c a e -1 172^ ^ ^ ^^Figure 3.3 Effect of 1% (wt/vol) aqueous suspensions of KC on the viability of various ESKAPE strains Dotted line at log10 =1 of Y axis represents the limit of detection for CFUs.  CFUs were determined at 0 h, 5 h, and 24 h of incubation for all strains and also at 48 h for E. faecium strains. ^ indicates that no viable cells could be recovered at that time point. Error bars represent the standard error (SE) of the mean of at least three independent replicates of each strain in these six groups (Behroozian et al. 2016).  E F D Chapter 3: Antimicrobial Properties of Kisameet Clay  64   Burkholderia cepacia complex, P. aeruginosa, and S. maltophilia isolated from cystic fibrosis patients 3.3.4.2.1 Cystic fibrosis    Recalcitrant chronic bacterial infections in humans represent a significant therapeutic problem worldwide whereby repeated challenge with antibiotics promotes the acquisition of drug-resistant bacteria both by providing a selection for infections with intrinsically resistant organisms and through selective pressure on existing organisms further elevating their resistance (Grant and Hung 2013). Cystic fibrosis (CF) is the most common lethal, heritable disorder among Caucasians, affecting 1 in 2,500 newborns is one such condition (Guggino and Stanton, 2006). Affected individuals suffering from mucosal immunodeficiency are vulnerable throughout their lives to chronic and ultimately deteriorating multi-year lung infections, account for most morbidity and mortality (over 90%) associated with CF (Grant and Hung 2013; Leitão et al. 2010). Patients with CF are susceptible at a young age to a range of MDR opportunistic Gram-negative bacteria such as P. aeruginosa, B. cepacia complex (BCC), and S. maltophilia, among which members of the BCC are particularly virulent pathogens (Ratjen et al. 2015; Davies 2002).        Eradication of these organisms from CF patients with current antimicrobial options is a major challenge. Thus, novel therapeutic approaches are urgently required. Overall, pulmonary infections caused by BCC in CF patients are associated with unpredictable rates of lung function decline, poorer prognosis, longer periods of hospitalization, and elevated death rates, especially among patients with more advanced pulmonary exacerbation or who have received lung transplantation (Mahenthiralingam 2005).       The BCC comprises at least 20 phenotypically similar, closely-related bacterial species which, over the past thirty years, have become recognized as highly problematic opportunistic pathogens in immunocompromised patients, most notably among people with CF and chronic granulomatous disease (CGD) (Mahenthiralingam 2005; Miller et al. 2015; Speert 2002). Their pathogenic responses in CF patients is not fully understood, but is likely due to multiple factors including high levels of both intrinsic Chapter 3: Antimicrobial Properties of Kisameet Clay 65 and acquired mechanisms of resistance to antimicrobials and known high risk of inter-patient transmissibility  (Speert 2002). They have been associated with adverse clinical courses, ranging from mild asymptomatic carriage to a fulminant decline in pulmonary function, necrotizing pneumonia, and septicemia known as “cepacia syndrome” (Zlosnik et al. 2015). The ability of BCC to evade the inhibitory action of multiple classes of antibiotics is a capacity at least partially accountable for the serious nature of infections caused by BCC bacteria (Rhodesa and Schweizera 2016). Eradication of these major pathogens with the limited availability of antimicrobial therapies is challenging, therefore new therapeutic strategies are urgently required (Ratjen et al. 2015; Rhodesa and Schweizera 2016). Indeed the high level of intrinsic antimicrobial resistance possessed by BCC represents a particularly difficult challenge to any novel broad-spectrum antimicrobial agents.      This study showed that KC has potent broad-spectrum antibacterial activity against the ESKAPE pathogens (Behroozian et al., 2016). To investigate whether KC may have value in the treatment of the range of serious bacterial pathogens involved in chronic pulmonary infections in CF patients, we conducted a study on 17 clinical pathogens consisting of twelve B. cepacia complex, four P. aeruginosa, and one S. maltophilia isolated from CF patients (Table 3.2). 3.3.4.2.2 Antibacterial resistance patterns of CF clinical isolates      Antibacterial susceptibility profiles of isolates were determined using a panel of 34 antibiotics (Table 3.3). All B. cenocepacia, B. cepacia, and B. stabilis isolates were extensively-drug resistant (XDR) (Magiorakos et al. 2012), whereas widespread multidrug resistance was observed among the other isolates (Table 3.7). All isolates exhibited resistance to first- and second-generation cephalosporins, ertapenem, meropenem, amoxicillin-clavulanic acid, ampicillin, and nitrofurantoin. All B. cenocepacia, B. cepacia, B. multivorans VC5602, and S. maltophilia isolates were resistant in addition to all seven aminoglycosides tested, while P. aeruginosa strains were also resistant to cefixime, cefpodoxime, sulfamethoxazole-trimethoprim, and trimethoprim. Collectively, a few isolates were resistant to ceftazidime and piperacillin. Chapter 3: Antimicrobial Properties of Kisameet Clay 66 Sequential isolates of B. cenocepacia and B. multivorans showed some differences in their resistance profiles. 67 Table 3.7 Resistance patterns of CF isolates to different classes of antimicrobial agents Filled black circles indicate resistance (inhibition ≤ 2mm from the edge of the disks of antibiotic); no mark indicates wider zone of inhibition  (at least three replicates for each antibiotic).  Spectinomycin is an aminocyclitol antibiotic, but as it is structurally related to the aminoglycosides, often considered alongside this group of antibiotics (Veyssier and Bryskier 2005; Ramirez and Tolmasky 2010). The amount (micrograms) per disk of antibiotics (Oxoid, BBL) is indicated in parentheses.   a, b, c  sequential isolates from the same patients that were previously evaluated for strain type by random amplified polymorphic DNA analysis (Speert et al. 2002b).  d two morphotypes of the strainVC15184: (-1, mucoid and -2, non- mucoid strain which was recovered during the course of this study and confirmed to have the same RAPD phenotype). The remaining strains were independent isolates from one patient. Amikacin (10)Gentamicin (30)Kanamycin (30)Neomycin (30)Spectinomycin (100)Streptomycin (10)Tobramycin (10)Ertapenem (10)Imipenem (10)Meropenem (10)Cephalothin (30)Cephazolin (30)Cefotetan (30)Cefoxitin (30)Ceftazidime (30)Cefixime (5)Cefpodoxime (10)Ceftriaxone (30)Cefotaxime (30)Amoxicillin-clavulanic acid (30)Ampicillin (10)Piperacilin (100)Colistin (10)Polymyxin B (300) Ciprofloxacin (5) Levofloxacin (5) Nalidixic acid (30)Sulfamethoxazole- trimethoprim (25)Sulfadiazine (250)Trimethoprim (5)Doxycycline (30)Tetracycline (30)Chloramphenicol (30)Nitrofurantoin (300)Sulfonamides TetracyclinesAminoglycosides Carbapenems 1st, 2nd, 3rd generation cephalosporins Penicillins Polypeptides QuinolonesIsolate Strain1 Burkholderia cepacia VC9490 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●2 Burkholderia cenocepacia C3921a ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●3 Burkholderia cenocepacia C8963a ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●4 Burkholderia cenocepacia C9343a ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●5 Burkholderia cenocepacia VC13195b ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●6 Burkholderia cenocepacia VC15185b ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●7 Burkholderia cenocepacia VC15442b ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●8 Burkholderia dolosa VC14902 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●9 Burkholderia multivorans VC5602c ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●10 Burkholderia multivorans VC16929c ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●11 Burkholderia stabilis VC7909 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●12 Burkholderia vietnamiensis VC9237 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●13 Pseudomonas aeruginosa VC8263 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●14 Pseudomonas aeruginosa VC15184-1d ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●15 Pseudomonas aeruginosa VC15184-2d ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●16 Pseudomonas aeruginosa VC17829 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●17 Stenotrophomonas maltophilia VC13512 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●Chapter 3: Antimicrobial Properties of Kisameet Clay  68  3.3.4.2.3 Antibacterial activity of KC suspensions and aqueous leachates against MDR clinical pathogens isolated from CF patients      The effect of KC against these clinical isolates was investigated in a viability assay using 1 or 10% (wt/vol) aqueous suspensions. KC exhibited potent in vitro antibacterial activity against a collection of MDR and XDR clinical isolates of BCC, P. aeruginosa, and S. maltophilia isolated from patients with CF. Exposure to KC reduced, and in most cases eliminated, the viability of all isolates tested (Fig. 3.4). After 24 h treatment of BCC strains with 10% KC suspensions, no viable cells could be recovered except for B. dolosa and B. multivorans VC5602, which required up to 48 h (Fig. 3.4B, C) and B. cepacia and B. cenocepacia C3921 that showed a 3-5 log10 decline in CFU in the same period of treatment (Fig. 3.4A, B). While a 1% suspension of KC did not inhibit BCC isolates completely (data not shown), this concentration of KC caused complete loss of viability of P. aeruginosa VC8263, VC15184-1, and VC17829 in 5 h and P. aeruginosa VC15184-2 and S. maltophilia within 24 h of treatment (Fig. 3.4D, E). In contrast, in controls without KC, the decline in CFU during the same periods of incubation was < 1 log10 for all isolates tested.      B . c e p a c iaT im e  (h )Log10 CFU/mL02448012345678              B . c e n o c e p a c iaT im e  (h )Log10 CFU/mL02448 02448 02448 02448 02448 02448012345678C 3 9 2 1C 8 9 6 3C 9 3 4 3V C 1 3 1 9 5V C 1 5 1 8 5V C 1 5 4 4 2^ ^ ^ ^ ^^^ ^ ^ ^  VC9490 B A 69 O th e r B u r k h o ld e r ia  s p .T im e  (h )Log10 CFU/mL02448 02448 02448 02448 02448012345678B . d o lo sa V C 14 9 02B . m u lt iv o ra n s V C 56 02B . m u lt iv o ra n s V C 16 9 29B . s ta b ilis V C 79 09B . v ie tn a m ie n s is V C 92 37^ ^ ^ ^^ ^ ^ ^P . a e r u g in o s aT im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524012345678V C 8 2 6 3V C 1 5 1 8 4 -1V C 1 5 1 8 4 -2V C 1 7 8 2 9^ ^ ^ ^ ^ ^^S . m a lto p h il iaT im e  (h )Log10 CFU/mL0 524012345678^Figure 3.4 Effect of aqueous suspensions of KC on the viability of CF isolates: 10% (wt/vol) against B. cepacia complex isolates (A-C) and 1% (wt/vol) against P. aeruginosa isolates (D) and S. maltophilia (E) CFUs have been determined at 0 h, 5 h, 24 h, and 48 h of incubation. Dotted line at log10 =1 of Y axis represents the limit of detection for CFUs.  ^ indicates that no viable cell could be recovered at that time point. Error bars represent the standard error (SE) of the mean of three independent replicates of each strain.  C D E VC13512Chapter 3: Antimicrobial Properties of Kisameet Clay 70      To identify the bactericidal effect of the soluble ions released from KC particles, a water-leachable fraction of the KC suspension was prepared and assayed for activity against these isolates. KC aqueous leachate (L100) was obtained as described previously in section 3.2.3. Although treatment of all strains with L500 was completely bactericidal after 5 h of incubation (data not shown), strains exhibited different sensitivity to L100 as shown in Fig. 3.5. A KC aqueous leachate (L100) was cidal to all the isolates. B. dolosa VC14902, B. stabilis VC7909, B. vietnamiensis VC9237, S. maltophilia VC13512 and all P. aeruginosa strains exhibited complete loss of viability after 24 h of treatment with L100 (Fig. 3.5C-E), while the same bactericidal effect took 48 h for B. cenocepacia C9343 and B. multivorans VC5602 (Fig. 3.5B, C). B. cepacia VC9490 exhibited more than 5 log10 decline in CFU during the period of treatment and all the B. cenocepacia strains, other than C9343, and also B. multivorans VC16929 exhibited ~1 to 4 log10 decline in viability (Fig. 3.5A-C). B . c e p a c iaT im e  (h )Log10 CFU/mL02448012345678B . c e n o c e p a c iaT im e  (h )Log10 CFU/mL02448 02448 02448 02448 02448 02448012345678C 3 9 2 1C 8 9 6 3C 9 3 4 3V C 1 3 1 9 5V C 1 5 1 8 5V C 1 5 4 4 2^B A VC9490 71 O th e r B u r k h o ld e r ia  s p .T im e  (h )Log10 CFU/mL02448 02448 02448 02448 02448012345678B . d o lo sa V C 14 9 02B . m u lt iv o ra n s V C 56 02B . m u lt iv o ra n s V C 16 9 29B . s ta b ilis V C 79 09B . v ie tn a m ie n s is V C 92 37^ ^ ^^ ^ ^ ^P . a e r u g in o s aT im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524012345678V C 8 2 6 3V C 1 5 1 8 4 -1V C 1 5 1 8 4 -2V C 1 7 8 2 9^ ^ ^^ S .  m a lto p h il iaT im e  (h )Log10 CFU/mL0 524012345678^^Figure 3.5 Effect of KC-L (L100) on the viability of B. cepacia complex isolates (A-C), P. aeruginosa isolates (D), and S. maltophilia (E)  Effect of aqueous leachate of KC (L100) on the viability of B. cepacia complex isolates (A-C), P. aeruginosa isolates (D), and S. maltophilia (E).  CFUs have been determined at 0 h, 5 h, 24 h, and 48 h of incubation. Dotted line at log10 =1 of Y axis represents the limit of detection for CFUs. ^ indicates that no viable cell could be recovered at that time point. Error bars represent the standard error (SE) of the mean of three independent replicates of each strain.  C D E VC13512Chapter 3: Antimicrobial Properties of Kisameet Clay 72 Antibacterial activity of KC against M. marinum as a model of M. ulcerans M. marinum, causing infections in fish and human, was used as a model for M. ulcerans (Hashish et al.2018). Growth inhibition assays using 250 mg mL-1 KC in 7H9 broth supplemented with 1% OADC and 0.2% glycerol showed complete eradication of M. marinum M strain within 2 days of treatment in vitro. The same inhibitory effect was observed on 7H10 agar media supplemented with 1% OADC and 250 mg mL-1 KC. 0 2 512 0 2 51201234567891 0M . m a r in u mT im e  (d a y )Log10 CFU/mLControlKC^ ^ ^Figure 3.6 Antibacterial activity of KC against M. marinum M. marinum M strain was used as a model for M. ulcerans. Mid-exponential phase growth of bacteria in 7H9 brothsupplemented with 1% OADC and 0.2% glycerol was treated with 250 mg mL-1 KC and incubated at 30˚C. Theinhibitory activity was studied during 12 days by determining the viability of bacteria treated with KC compared tothe control without treatment.Dotted line at log10 =1 of Y axis represents the limit of detection for CFUs. ^ indicates that viable cells were below the limit of detection at that time point.  Error bars represent the standard error (SE) of the mean of at least three independent replicates.    Antifungal activity of KC suspensions against C. albicans and C. neoformans      To extend the application of KC to other clinically-relevant microbes, its activity was tested against two major pathogenic fungal species. C. albicans is the most prevalent human fungal pathogen which naturally resides as a constituent of the healthy microbiota; however, it can overgrow as an opportunistic Chapter 3: Antimicrobial Properties of Kisameet Clay 73 pathogen and cause superficial and mucosal infections and/or disseminate into the bloodstream and cause invasive infections in internal organs (Kim and Sudbery 2011; Poulain 2015). While a 1% aqueous suspension of KC  inhibited C. albicans at 37˚C but not completely at 30˚C (data not shown), 5% (wt/vol) suspensions of KC completely eradicated C. albicans below the limit of detection within 24 h of treatment at both temperatures (Fig. 3.7A). In addition, the activity of KC was tested against C. neoformans, an emerging encapsulated fungal pathogen which infects both immunocompromised and immunocompetent individuals, causing diseases ranging from cutaneous lesions to more systemic and possibly fatal diseases such as pulmonary and meningeal infections (Shirley and Baddley 2009; Schmalze et al. 2016; Srikanta et al. 2014). KC suspensions showed fungicidal activity against C. neoformans (Fig. 3.7B), reducing the CFUs below the detection limit within 24 h of treatment. Together, these results illustrate the broad-spectrum antimicrobial activities of KC. Figure 3.7 Effect of 5% (wt/vol) aqueous suspensions of KC on the viability of fungal strains, C. albicans SC5314 (A) and C. neoformans H99 (B) Suspensions of exponential-phase fungal cells were suspended in 5% (wt/vol) aqueous suspensions of KC. At the indicated time points, CFUs were enumerated by dilution and plating. Dotted line at log10 =1 of Y axis represents the limit of detection for CFUs. ^ indicates that viable cells were below the limit of detection at that time point.  Error bars represent the standard error (SE) of the mean of at least three independent replicates.     A B Chapter 3: Antimicrobial Properties of Kisameet Clay 74 Discussion      Antibiotics have revolutionized modern medicine and saved countless lives; however, the rapid emergence of resistant strains worldwide threatens a return to the pre-antibiotic era (Crofts et al. 2017). As bacterial evolution toward resistance has overwhelmed traditional antibiotic repertoires, natural alternatives are receiving increasing attention for their potential antimicrobial applications (Lewis 2012). Although natural products have been an inexhaustible source of novel therapeutic agents and bioactive compounds (Davies 2010), they require rigorous scientific analysis to support their efficacy and define their complex nature and active principal components.       This study presents a naturally-occurring clay mineral, KC, with potent and broad-spectrum antimicrobial properties in vitro. The antimicrobial assays described in this chapter confirm the potent inhibitory activity of KC against a variety of bacterial and fungal pathogens. Moreover, aqueous extracts of KC (without mineral particles) demonstrate the same broad-spectrum antibacterial activity, suggesting that the active component(s) can be extracted and used in defined preparations. In addition to its antibacterial activity, KC exhibits antifungal activity against C. albicans and C. neoformans, two major pathogenic fungi mostly causing infections in immunocompromised patients (Kim and Sudbery 2011; Shirley and Baddley 2009). Modern medicine has paradoxically increased the number of immunocompromised patients who are more vulnerable to fungal infections named as “hidden killers” (Kim 2016; Brown et al. 2012). These two fungal pathogens exhibited increased susceptibility to KC under 37˚C compared to 30˚C. The temperature-related sensitivity to antimicrobial therapy has been reported previously (Odds 1993; Pettit et al. 2010).       Among the different bacterial strains tested, Gram-negative bacteria were more sensitive to both KC aqueous suspensions and leachates than Gram-positive bacteria. S. aureus exhibited lower susceptibility to KC leachates with a prolonged time for complete elimination of cells and also low tolerance to low-pH buffers (Valero et al. 2009). The broad-spectrum antibacterial activity of KC leachates suggests that the soluble/exchangeable fraction of KC is involved in the activity as reported for other clay minerals Chapter 3: Antimicrobial Properties of Kisameet Clay 75 (Cunningham et al. 2010; Otto et al. 2010; Morrison et al. 2016). Otto et al. (2010) reported the antibacterial activity of aqueous leachates of two natural antibacterial clay mixtures and demonstrated that the in vitro antibacterial activity of the natural clay may depend on chemical desorption of specific metal ions from the surface of the clay particles.        Previous studies with certain antibacterial clays, CsAg02 and OMT, suggested a role for exchangeable cations in their bactericidal activity (Williams and Haydel 2010, Morrison et al. 2014). Cunningham et al. demonstrated that mineral clays exhibiting cidal activity contained higher concentrations of chemically accessible metal ions than non-antibacterial samples and that their antibacterial activity was due to the release of exchangeable metal ions in a pH-dependent manner (Cunningham et al. 2010). Additional studies using speciation modeling and cation supplementation have indicated the specific roles of divalent metal ions (Fe, Cu, and Zn) in the antibacterial activity of certain clay minerals (Otto and Haydel 2013a). Elemental analysis revealed that KC aqueous leachates contain considerable amounts of metal ions such as Al and Fe, as reported previously (Hauser 1952; Williams et al. 2011). Different metal ions cause a variety of damage to microbial cells as a consequence of membrane function impairment, interference with nutrient assimilation, production of oxidative stress, by producing reactive oxygen species and depleting antioxidants, and protein dysfunction and enzyme inactivation (Lemire et al. 2013).       The ESKAPE pathogens are responsible for the majority of recalcitrant bacterial outbreaks in nosocomial settings, but the therapeutic choices are extremely limited (Santajit and Indrawattana 2016).  They are predicted to be of increasing relevance in infectious disease for the foreseeable future, but the current antibiotic armamentarium has little to offer in terms of treatment, and there are few novel antimicrobial agents under development that show promise in relieving the health crisis caused by these organisms (Pendleton et al. 2013).      Despite intensive searches for new antimicrobial agents, there are few active candidates in the pipeline. KC exhibits bactericidal effects against a panel of both Gram-positive and Gram-negative MDR ESKAPE strains (Behroozian et al. 2016). Although there are differences in susceptibility between Chapter 3: Antimicrobial Properties of Kisameet Clay 76 isolates of the same species, to date no resistance to KC has been observed. These results suggest that the broad-spectrum antibacterial activity of KC may have potential for the treatment of ESKAPE infections, especially in last-resort situations. Such ancient medicinals and other natural mineral-based agents may provide new weapons in the battle against multidrug-resistant pathogens. Harrison et al. (2015) described a successful application of an ancient natural medicine. Therefore, reassessment of the potency and mechanisms of action of natural agents deserves more attention.     Early and persistent lung infections represent a major therapeutic challenge in CF, which accounts as the major determinant of life span in affected patients (Speert et al. 2002b; Davies 2002). BCC bacteria are responsible for the most challenging of all pulmonary infections in CF patients with unpredictable clinical outcome, poor prognosis, limited effective antimicrobial options, and high mortality rate (Zlosnik et al. 2002; Grant and Hung 2013; Ratjen et al. 2015). Due to their high level of antibiotic resistance, current antimicrobial options for the therapy of BCC infections are limited and eradication of the organisms from CF patients is a major challenge. Thus, novel therapeutic approaches are urgently required (Rhodesa and Schweizer 2016).       This study elucidates the in vitro antibacterial effect of KC against a panel of MDR and XDR clinical isolates from CF patients, including P. aeruginosa isolates, as the most common pathogen in CF patients (Zlosnik et al. 2015), as well as sequential isolates from chronic infections of B. cenocepacia and B. multivorans, the two most clinically relevant  BCC species  accounting for around 85% of all BCC infections (Rhodesa and Schweizera 2016; Tegos et al. 2012), and S. maltophilia, as well as other BCC strains. These results extend our previous observations on the potent bactericidal effect of KC against MDR clinical isolates of P. aeruginosa among ESKAPE pathogens. Notably, P. aeruginosa VC15184-1, a mucoid strain, and its non-mucoid derivative VC15184-2 were similarly affected by KC, indicating that a mucoid phenotype in P. aeruginosa may not be a major factor in resistance to KC. These studies further expand the spectrum of activity of KC to include isolates of some of the most challenging pathogens from Chapter 3: Antimicrobial Properties of Kisameet Clay 77 chronic infections and suggest that further studies of other globally important Burkholderia pathogens such as B. pseudomallei should be implemented (Limmathurotsaku et al. 2016).        The most challenging problem in the management of CF is the early development of chronic infections, which requires successful colonization followed by long-term survival of the pathogen (Leitão et al. 2010; Mahenthiralingam et al. 2005). KC may be a valuable potential therapeutic option as a complementary or a suppressive antimicrobial treatment for pathogenic colonization and chronic pulmonary infections in CF patients and also in cases of CGD. Recently, the successful application of a novel inhalation antibiotic therapy to deliver high concentrations of medicine to the lung has been reported (Ratjen et al. 2015). KC aqueous leachate has the potential to be developed and studied for aerosolized drug administration. Further detailed in vivo studies using animal models and cytotoxicity investigations as well as studying the potential targets need to be carried out. These studies may offer novel solutions for suppressive treatment of pathogenic colonization and chronic pulmonary infections.          Such studies provide a strong argument for the application of KC in the treatment of infectious diseases as well as a better understanding of the antibacterial properties of the material. My studies can provide a source for defining and controlling the antibacterial activity of KC and could lead to clay derivatives appropriate for clinical applications. In addition, as natural clay minerals are heterogeneous mixtures, defining the specific characteristics needed for antibacterial activity is essential to standardize and improve chemical consistency for medicinal applications. Continued studies on the role of metal ions are needed to reveal the mode(s) of action and identify principal active components. Collectively, the potent antibacterial action of KC against human pathogens of clinical importance together with its antifungal activity emphasize that KC could have applications as a natural antimicrobial agent for use against topical infections caused by antibiotic-resistant microbes.       Identification of the lethal mode(s) of action of KC should allow the development of more defined, homogeneous, consistent, and stable preparations of KC for therapeutic applications. Furthermore, as antibacterial mineral clays exhibit considerable variation in their mineralogical and chemical Chapter 3: Antimicrobial Properties of Kisameet Clay 78 compositions, physico-chemical properties, and pH buffering capacities, elucidating the mechanism(s) will not only provide insight into what makes medicinal clay minerals antibacterial but will also guide in the development of natural mineral-based alternative antimicrobial agents for a variety of therapeutic applications. 79  Roles of pH and Metal Ions in the Antibacterial Activity of KC Introduction      Certain metals are critical components in nearly all aspects of microbial metabolism, growth, and differentiation with diverse structural and functional roles (Gadd 1992; Bruins et al. 2000). While metals and metalloids dominate the periodic table, a minority, such as Ca, Co, Cu, Fe, Mg, Ni, and Zn are required elements and recognized as essential metals; whereas some (i.e. Ag, Al, Cd, Hg, and Pb) are xenobiotic, with no known nutrient value or essential biological role (Ji and Silver 1995; Lemire et al. 2013; Tamás et al. 2014). Essential metals function as integral catalysts for biochemical reactions, as key structural elements and stabilizers of biomolecules, or in maintaining osmotic balance (Gadd 1992). Metals are essential in the structures of nearly half of the known proteins, or as trace elements playing vital roles in key cellular processes (Lemire et al. 2013). In fact, many central biochemical and bioenergetic cellular processes including the synthesis of biomolecules, electron transport chain, signal transduction, catalysis, and  cell division depend entirely on metal-ion cofactors (Waldron and Robinson 2009; Chandrangsu et al. 2017; Weiss and Carver 2018). For instance, essential metals like Fe, Cu, and Ni are involved in redox processes, Mg and Zn stabilize DNA and various enzymes via electrostatic forces, and Na and K regulate intracellular osmotic pressure (Nies 1992; Bruins et al. 2000). Due to the reactive nature of metal ions, their catalytic properties, and high affinity for amino acids, microorganisms try to tightly maintain their total intracellular and exchangeable-metal pools within the cell at defined concentrations to inhibit the detrimental consequences of inappropriate metalation or intoxication (Loutet et al. 2016; Imlay 2015).      While essential metals are lethal to cells by acting as abiotic microbicides at excessive amounts, certain nonessential metals and metalloids (i.e. Ag, Hg, and Te) exert acute toxicity toward bacteria at extremely low concentrations, (Nies 1999; Lemire et al. 2013). Major mechanisms of toxicity arise generally due to strong coordinating properties via the formation of unspecific complex compounds, the Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 80 substitution or displacement of essential metal ions from their native cellular sites, the stability of metal-biomolecule complexes formed, or by blocking the functional groups of key biomolecules (Gadd 1992; Hobman and Crossman 2015). Moreover, nonessential metals bind irreversibly with higher affinity to thiol-containing groups and oxygen compared to essential metals (Bruins et al. 2000). Metal speciation rather than total concentration is critical to its bioavailability and the subsequent interactions with bacteria (Lemire et al. 2013). Recent studies show that different transition metals can cause distinct and discrete types of damage to microbial cells (Santo et al. 2011; Sun et al. 2011; Azam et al. 2012, Tran et al. 2010). Depending on the metal, intoxication alters the conformation of proteins and nucleic acids, denatures enzymes and abolishes their specificity, and interferes with oxidative phosphorylation and osmotic balance (Lemire et al. 2013). Collectively, metal toxicity has been attributed to protein dysfunction, impaired cell membrane integrity, interference with nutrient assimilation, generation of reactive oxygen species (ROS) and depletion of antioxidants, or genotoxicity (Gadd 1992; Bruins et al. 2000; Lemire et al. 2013).      The potent antimicrobial properties of metals have been employed throughout the history of medicine (Vaidya et al. 2017). Aside from plants and other traditional medicaments which have been used therapeutically to heal infectious diseases since antiquity, specific toxic metals such as Hg and As were exploited as the first extensively developed and applied antimicrobial agents (Abrams and Murrer 1993; Miao et al. 2011). In fact, Salvarsan, an organoarsenic compound, was the first modern chemotherapeutic agent with effective applications (Fig. 1.1; Abrams and Murrer 1993). The historical application of metal-based compounds as antimicrobials stretched into the 20th century until it was diminished upon the introduction of antibiotics and organic antimicrobial compounds in the mid-20th century (Turner 2017; Gugala and Turner 2018). Nowadays, with the escalating threat of MDR superbugs and the dearth of novel antimicrobial agents in the pipeline, the application of metal-based compounds with potent antimicrobial activity is undergoing a renaissance. Metal-based antimicrobials (MBA) such as metallic surfaces and coatings, nanomaterials, and chelates have a multitude of applications in health care, Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  81  agriculture, and industry (Kollef et al. 2008; Lemire et al. 2015; Turner 2017, Gold et al. 2018). Various metal compounds, in particular those containing Ag, Cu, and Zn, metal-based nanoparticles, and products impregnated with these metal ions have been used extensively as microbicides (Gold et al. 2018; Santo et al. 2011; Sun et al. 2011; Da Silva Martins et al. 2018; Azam et al. 2012, Tran et al. 2010).        Metal-bearing clay minerals are ubiquitous in nature (Stucki 1996; Williams et al. 2009). Al and Fe are the two most abundant metals in the earth’s crust, while clays constitute the most important reservoir of these metals (Gadd 2010; Bojić et al. 2002; Stucki 2013; Vorhies and Gaines 2009). Clay minerals adsorb transition metals to their surface due to their net negative charge, and in a hydrated environment exchange these cationic species with the surroundings, depending on the ionic strength of the aqueous medium and cation selectivity of the clay (Williams et al. 2011; Otto and Haydel 2013). Most antibacterial clay minerals originate from hydrothermally-altered volcanics where volcanogenic fluids created minerals harboring reduced metal ions (Williams 2017). Studies with other clays suggest that exchangeable transition metal ions such as Fe, Zn, and Cu, inherent in natural clay minerals, may underlie their antibacterial activity and also have been exploited to make metal-ion exchange-based antibacterial clays (Cunningham et al. 2010; Otto and Haydel 2013a; Morrison et al. 2016). Moreover, the presence of pyrite (FeS2) in some clay minerals has been reported to be important for bactericidal action (Williams et al. 2011; Morrison et al. 2014).       With the characterization of KC broad-spectrum antimicrobial activities against MDR and XDR clinical pathogens, the principal goal of this research is to elucidate the mechanism(s) of antibacterial action. Elemental analyses of KC mineral have revealed that this clay contains large amounts of Al and transition metals, particularly Fe, Cu, and Zn (Table 2.3). As certain metal ions are more soluble at low pH, the acidic nature of KC leachates may be required for their activity. Collectively, a hypothesis is that the antibacterial activity of KC is due to the pH-dependent release of exchangeable transition metal ions (especially Fe), generation of ROS, and impairment of bacterial cell membrane integrity. Thus, the research aims will be to: (a) evaluate the role of exchangeable transition metal ions, particularly Fe, in the Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  82  antibacterial activity of KC, (b) elucidate the role of oxidative stress in the bactericidal activity of KC, and (c) study whether cell membrane integrity is impaired upon treatment with KC.        The goal of this chapter is to evaluate the influence of low-pH on the bactericidal activity of KC leachates and to determine the role of metal ions. To investigate the role of soluble metal ions, a variety of cation chelators, chemical analyses, and metal solutions will be used. These studies may permit the identification of principal component(s) involved in the bactericidal activity of KC. Elucidating their roles will aid in studying the second and third aims.   Materials and methods  Bacterial strains and growth conditions       E. coli MG1655, S. aureus RN4220, and P. aeruginosa PAO1 were used as described in section 3.2.1.  E. coli MG1655 and P. aeruginosa PAO1 were grown in LB broth or on LB agar and S. aureus RN4220 was grown in TSB or on TSA.  Chelation assays  Treatment of KC suspensions with chelating agents       To study the role of metal ions, KC mineral and aqueous leachates were treated with three chelating agents as described by Cunningham et al. (2010) with some modifications. Briefly, KC aqueous suspensions were prepared as described in section 3.2.3. KC mineral (1g dry clay per 20 mL, 5% wt/vol) was washed with cation chelator solutions (10 or 100 mM aqueous ethylenediaminetetraacetic acid (EDTA, Sigma), 10 mM aqueous 2,2ˈ-bipyridyl (BPY, Sigma); (final concentrations), or dH2O with continuous shaking for 24 h at room temperature. KC particles were then washed twice with dH2O to remove remaining chelators and collected by centrifugation (25,000 rpm for 2 h), dried, ground using a mortar and pestle, and autoclaved for 1 h at 121˚C. To treat KC with deferoxamine mesylate (DFO, Sigma), a KC mineral suspension (5% wt/vol) was washed twice with DFO (10 mM final concentration), or dH2O with continuous shaking for 3 h at room temperature. KC particles were then washed twice with Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  83  dH2O to remove remaining chelator and collected as described previously. The inhibitory activity of aqueous suspensions (1% wt/vol) of the treated KC samples compared to the original clay was investigated in an antibacterial assay against E. coli MG1655 as described in section 3.2.6.1.   Treatment of KC leachates with chelating agents      To determine the role of soluble cations in the antibacterial activity, a KC aqueous leachate (L50) was prepared as described in section 3.2.3 and an EDTA solution was added to a final concentration of 10 mM. The pH of EDTA-treated leachate was measured. As this treatment resulted in an increase of pH to 7.08, a sample of L50 containing EDTA was subsequently adjusted with 1 M HCl to a pH similar to the initial acidic pH of L50 (4.14) and then sterilized through a 0.22 μm filter. In addition, a BPY solution was added directly to L50 to a final concentration of 1 mM. As treatment with BPY changed the pH of L50 from 4.14 to 5.0, a sample of leachate containing BPY was subsequently adjusted with 1 M HCl to a pH similar to the initial pH of L50 and then sterilized by filtration through a 0.22 μm membrane. All leachates were tested for their comparative antibacterial activity. Moreover, DFO was added to a final concentration of 1 mM to L50 and the pH was measured. As this treatment reduced the pH to 3.4, a sample of the DFO-treated leachate was subsequently adjusted with 1 M sodium hydroxide (NaOH) to generate a pH environment similar to the initial acidic pH of the L50. The antibacterial activity of KC leachate was compared to the DFO-treated L50 in a viability assay against E. coli as described.         pH adjustment of aqueous leachates and precipitation assays       To investigate the role of pH in KC leachate activity, a KC aqueous leachate (L50) was prepared as previously described. The initial pH was measured and a sample of the leachate was gradually adjusted to pH 7.0 using 1 M NaOH. The leachate was then sterilized by filtration through a 0.22 μm membrane (Millipore) before antimicrobial testing. Moreover, in another study, the pH of KC leachates (L50 and L100) was adjusted to 7.0 using 1 M NaOH. The leachates were centrifuged at 10,000 rpm for 20 min and the supernatant was carefully collected from the top and the precipitates were resuspended in the same Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 84 amount of dH2O as their original volumes. The pH of supernatants and the resuspended precipitates were measured and then a sample of each was adjusted to the original pH of KC leachates, using 1 M HCl and filter-sterilized. The antibacterial activity of the leachates and their derivatives were compared to the initial leachates by testing in a viability assay with E. coli as described previously in section 3.3.2. Elemental analysis by ICP mass spectrometry (ICP-MS)         Measurements of the elemental concentrations in the KC leachates and their derivatives were determined using ICP-MS. The accuracy and precision of the analytical method were monitored by analysis of a certified reference material, Trace metals-clay 2 (CRM-2) (Sigma). Briefly, samples were digested in 1% nitric acid prior to analysis. Internal calibration standards were prepared from multi-element stock solutions (PerkinElmer Pure Plus; Inorganic Ventures). Indium (In) and scandium (Sc) at 100 ppb were also added as internal standards. Dilutions of 1:50 were analyzed along with undiluted samples and were calibrated to multi-element reference standards. ICP-MS was conducted using the PerkinElmer NexIon 300D ICP-MS instrument. As sulfur (S) is not efficiently ionized by ICP–MS, or shows interferences, S analysis was performed by ICP-OES as described previously in section 3.2.4 (Murray et al. 2000).    Preparation of MES- buffered defined minimal medium (MBMM)     To investigate the inhibitory action of leachates on growing bacteria, different media were tested (data not shown). The composition of media, especially the amount of carbon source, undefined components such as peptone or yeast extract, and buffering agents can significantly affect the antibacterial activity and in some cases mask the activity. A defined minimal medium, MES-buffered minimal medium (MBMM), was used for testing the antibacterial activity of KC leachates and metal solutions on the growing bacteria and was prepared as described by Rathnayake et al. (2013) with some modifications. This defined minimal medium has been formulated by considering the nutritional requirements of bacteria, to prevent excessive metal chelation, and to buffer the pH with 2-(N-morpholino)ethanesulfonic acid (MES) as a Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  85  Good’s buffer. The components (g L-1) of MBMM (pH 6.4) were as follows: MES, 1.95; Na2HPO4, 0.01; NH4Cl, 0.05; CaSO4, 0.14; FeSO4.7H2O, 0.004; KCl, 0.02; MgSO4.7H2O, 0.24; and 1 mL of SL7 trace element solution (Biebl and Pfenning 1981). SL7 element solution consists of the following components (mg L-1): CoCl2.6H2O, 200; CuCl2.2H2O, 20; H3BO3, 60; MnCl2.4H2O, 100; NaMoO4.2H2O, 40; NiCl2.6H2O, 20; ZnCl2, 70; and 1 mL of HCl (25%). 0.2% (wt/vol) glucose was also added to the medium as carbon source.       In present study, MBMM was supplemented with 0.01% (wt/vol) NZ- amine (a defined preparation of enzymatic digest of casein) (Sigma) for E. coli MG1655 and 0.1% NZ-amine for P. aeruginosa PAO1 based on the growth curve studies (data not shown). For S. aureus RN4220, in addition to 0.1% NZ-amine, MBMM was supplemented with vitamins (mg L-1) (Sigma) as follows: biotin (0.05), thiamine hydrochloride (1), nicotinic acid (1), pantothenic acid (1), pyridoxal hydrochloride (2), riboflavin (0.5), and pyridoxamine dihydrochloride (2).  Preparation of single metal ion solutions (FeCl2, FeCl3, AlCl3) and metal ion mixture (MIM)      Metal ion stock solutions were prepared by adding chloride salts (AlCl3, CoCl2.6H2O, CuCl2.2H2O, FeCl2.4H2O, FeCl3.6H2O, NiCl2.6H2O, and ZnCl2) (Sigma) to a final concentration of 100 mM for Al and Fe and 10 mM for Cu, Co, Ni, and Zn in sterile dH2O. All stock metal solutions except Fe solutions, which were prepared freshly before experiments, were passed through a 0.22 µm Millipore filter into sterile glass vials and stored at room temperature in the dark. Working solutions of single metals of Al3+, Fe2+ and Fe3+ were prepared by diluting the metal stocks in sterile dH2O to the concentrations found in KC-L00 by ICP-MS as described in Table 4.2. To obtain MIM, a mixture of Al3+, Co2+, Cu2+, Fe2+, Ni2+, and Zn2+ was prepared based on the reported concentrations by ICP-MS. The pH of each working single metal solution and MIM were adjusted to KC-L100 pH with 1 M NaOH. All the metal solutions were sterilized using a 0.22 µm filter prior to testing.  Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  86   Antibacterial activity of metal ion solutions on the viability and growth of bacteria      From overnight cultures of E. coli MG1655 and P. aeruginosa PAO1 in LB broth and S. aureus RN4220 in TSB, mid-exponential phase growth of strains were prepared for viability assays. Cells were then harvested by centrifugation, rinsed once in sterile PBS (pH 7.4), and resuspended in either KC-L (L100), in single metal ion solutions (FeCl2, FeCl3, and AlCl3), metal ion mixture (MIM), or in dH2O only (to study the viability of organisms in the absence of treatment) at an initial cell concentration of ~107 CFU mL-1. Suspensions were then incubated at 37˚C with shaking at 200 rpm. Viability was determined by 10-fold serial dilution and duplicate plating of aliquots removed at the start of experiments for initial CFUs on LB agar plates and following 5 and 24 h of exposure to KC-L or metal solutions.       Considering the crucial role of media formula in studying metal ion toxicity, growth inhibition assays were performed using a defined minimal medium, MBMM with the described supplementations. From overnight cultures of bacterial strains, the washed cells of mid-exponential phase growth were prepared and resuspended in MBMM broth at an initial cell concentration of ~107 CFU mL-1 and then treated with KC-L (L100), single metal solutions (FeCl2, FeCl3, and AlCl3), MIM, or dH2O in a (1:1) ratio. Suspensions were then incubated at 37˚C with shaking at 200 rpm and CFUs were determined by 10-fold serial dilution and duplicate plating of aliquots removed at the start of experiments and following 5 and 24 h of exposure to KC-L or metal solutions.   Determining minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) of metal ion solutions     MIC assays were performed based on CLSI broth microdilution method (CLSI 2015) and as described by Wiegand et al. (2008). Briefly, bacterial strains were cultured to mid-exponential phase as described previously. The bacterial cells were collected and washed with PBS (pH 7.4) once and resuspended in sterile deionized water to a concentration ~108 CFU mL-1 and then diluted 1:100 to obtain the bacterial inoculums. Single metal solutions of Al3+, Co2+, Cu2+, Fe2+, Fe3+, Ni2+, and Zn2+ were freshly prepared and Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  87  at least 10 concentrations of each metal were examined in triplicate. MIC assays were performed in sterile 96-well microtiter plates using MBMM defined minimal medium with described supplementations for each bacterial strain. Plates were incubated at 37°C with 150 rpm shaking for 24 h prior to determination of MICs. The MBCs were determined by plating 10 µL aliquot of each well on LB agar plates using the viable cell counting method. According to CLSI guidelines, MIC was defined as the lowest concentration of an antimicrobial agent that inhibits visible growth of an organism, while MBC defined as a ≥ 99.9% reduction in bacterial survival (CLSI, 1999). To assess the impact of media formula on MIC and MBC measurements, LB-Miller broth (tryptone, 5; yeast extract, 5; and NaCl, 10) LB-Lennox broth (tryptone, 10; yeast extract, 5; NaCl, 5) (g L-1 of dH2O), and LB-Miller broth without salt were prepared and comparative MIC and MBC of the single metal ion solutions were investigated.   Results  Effect of cation chelating agents on the antibacterial activity of KC mineral and leachate      Studies with other antibacterial clays suggested that cations may underlie the antibacterial activity of mineral clays (Cunningham et al. 2010; Otto and Haydel 2013a; Morrison et al. 2016). Therefore, to investigate the effect of various cation chelators to specifically sequester activity of KC, the broad-spectrum cation chelator EDTA was used. EDTA is noted for its ability to sequester a variety of metal ions, including Al, Ca, Cu, Fe, Mg, and Ni (Flora and Pachauri, 2010). As shown in Fig. 4.1A, pre-washing of dry KC with 100 mM EDTA eliminated the antibacterial activity of KC against E. coli. Although KC pre-washed with 10 mM EDTA exhibited reduced inhibitory activity (~4.5 log10) within 5 h of incubation, in comparison to KC washed with H2O alone, both 10 mM and H2O-treated KC reduced the bacterial viability below the detection limit within 24 h of treatment.        While this finding suggests that removal of metal ions might reduce KC activity, it is also possible that an increase in pH by EDTA treatment also influences the activity of the treated clay. Therefore, KC Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 88 aqueous leachates, whose pH could be more easily manipulated than suspensions, were treated with EDTA. This study showed that, as for the washed clay, antibacterial activity was eliminated by adding 10 mM EDTA (final concentration) directly to the aqueous leachate KC-L (Fig. 4.1B). Since adding EDTA to KC-L resulted in an increase in pH -from 4.14 to 7.08- (Table 4.1); the pH of KC-L treated with EDTA was adjusted to the initial low-pH of the leachate (4.14), which did not restore the activity. These results suggest that exchangeable divalent and trivalent cations are important components in KC toxicity.  0 4 8 1 2 1 6 2 0 2 4012345678E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mLdH2O ( 6.8)KC ( 4.3)H2O-treated KC ( 4.6)10 mM EDTA-treated KC ( 6.3)100 mM EDTA-treated KC ( 7.3)A Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 89 0 4 8 1 2 1 6 2 0 2 4012345678E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mL   dH2O ( 7.0)H2O ( 4.1)KC-L ( 4.14)KC-L + EDTA ( 7.1)KC-L + EDTA ( 4.1)Buffer ( 4.1)Figure 4.1 Viability of E. coli MG1655 in 1% (wt/vol) aqueous suspension of KC pre-washed with 10 or 100 mM EDTA (A) and KC-L (L50) treated with 1 mM EDTA and pH adjustment (B) The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. (pH values in parentheses)      As KC is an iron-rich clay (Hauser 1952; Svensson et al. 2017), and ICP analysis of KC bulk mineral and aqueous leachates showed significant amounts of Fe (Table 2.3; Table 3.4), the effect of two Fe chelators, BPY and DFO on KC activity were investigated. BPY is a bidentate chelator that predominantly binds Fe2+, while DFO is a hexadentate chelator that preferentially chelates Fe3+ (Elandalloussi et al. 2003; Makrlik and Van̆ura 1992; Dionis et al. 1991; Kontoghiorghes 1995). Pre-washing of dry KC with 10 mM BPY resulted in an ~1.5 log10 increase in the viability of E. coli compared to dH2O-treated KC within the first 5 h of incubation, but after 24 h, antibacterial activity was identical to that of non-treated clay (Fig. 4.2A). However, when 1 mM BPY (final concentration) was directly added to KC-L, the antibacterial activity decreased by 4 log10 compared to the original leachate and adjusting the pH to the initial value (4.14) did not restore activity (Fig. 4.2B).  B 90  0 4 8 1 2 1 6 2 0 2 4012345678E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mLdH2O ( 6.8)KC ( 4.3)H2O-treated KC ( 4.6)BPY-treated KC ( 4.8)T im e  (h )Log10 CFU/mL0 4 8 1 2 1 6 2 0 2 4012345678dH2O ( 7.0)KC-L ( 4.14)KC-L + BPY ( 5.0)KC-L + BPY ( 4.1)Buffer ( 4.1)H2O ( 4.1)E . c o li  M G 1 6 5 5Figure 4.2 Viability of E. coli MG1655 in 1% (wt/vol) aqueous suspension of KC pre-washed with 10 mM 2,2ˈ-bipyridyl (BPY) (A) and in KC-L (L50) treated with 1 mM BPY and pH adjustment (B). The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. (pH values in parentheses) A B Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 91      To examine the role of ferric iron in the antibacterial activity of KC, DFO, a strong Fe3+ chelator (stability constant ~30.6) (Table B.1) was used. Fig. 4.3A shows that pre-washing of dry KC with 10 mM DFO supported the viability of E. coli about 2 log10 compared to H2O-treated clay after 5 h of incubation. In addition, when L50 was supplemented with 1 mM DFO, the activity was almost eliminated (Fig. 4.3B), which together suggests a role for ferric iron in the activity of KC. However, as DFO is able to form strong complexes with other trivalent ions, notably Al3+ and Cr3+ (Day and Ackrill 1993; Keberle 1964) and chelate some of divalent cations such as Co2+, Cu2+, Fe2+, Ni2+, and Zn2+ with lower stability constants (Table B.1) (Smith 2013; Yokel 2002), this does not exclude roles of these cations in the bactericidal activity of KC.  0 4 8 1 2 1 6 2 0 2 4012345678E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mL   dH2O ( 6.8)dH2O + DFOKC( 4.3)H2O-treated KC ( 4.6)DFO-treated KC ( 3.3)A Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 92 0 4 8 1 2 1 6 2 0 2 4012345678E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mL   dH2O ( 6.8)dH2O + DFOKC-L ( 4.14)KC-L+ DFO ( 3.4)KC-L+ DFO ( 4.13)Figure 4.3 Viability of E. coli MG1655 in 1% (wt/vol) aqueous suspension of KC prewashed with 10 mM deferoxamine (DFO) (A) and in KC-L (L50) treated with 1 mM DFO and pH adjustment. The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates.     (pH values in parentheses) Table 4.1 pH comparison of KC aqueous suspensions and leachates treated with EDTA, 2,2ˈ-bipyridyl (BPY), or deferoxamine (DFO) KC  suspension preparation pH KC aqueous leachate preparation pH 1% (wt/vol) KC aqueous suspension 4.3 KC-L (L50) 4.14 H2O treated KC 4.6 KC-L + 10 mM EDTA 7.08 10 mM EDTA-treated KC 6.3 KC-L + 10 mM EDTA- adjusted pH 4.14 100 mM EDTA treated KC 7.3 KC-L + 1 mM BPY 5.0 10 mM BPY treated KC 4.8 KC-L + 1 mM BPY- adjusted pH 4.14 10 mM DFO treated KC 3.3 KC-L + 1 mM DFO 3.4 10 mM DFO treated KC- adjusted pH 4.2 KC-L + 1 mM DFO- adjusted pH 4.13  (pH values represent pH of 1% (wt/vol) aqueous suspensions of KC preparations (left) or KC aqueous leachates (KC-L) (right). B 93 Table 4.2 Elemental composition of KC leachates, L50, L100, and L500. Analyte levels were determined by ICP-MS Element L50 L100 L500  MDL     µg/L   µM    µg/L   µM    µg/L  µM ppb Ag 107 BDL - BDL - BDL - 1.48E+00 Al 27 7.45E+03 275.83 1.69E+04 624.14 9.67E+04 3580.89 4.59E+00 As 75 3.44E+00 0.04 3.21E+00 0.04 4.55E+00 0.06 1.49E+00 Ba 138 8.43E+00 0.06 1.38E+01 0.10 1.09E+01 0.08 1.50E-01 Be 9 5.36E+00 0.59 9.03E+00 1.00 3.68E+01 4.08 2.10E-01 Ca 44 6.55E+04 1488.24 1.26E+05 2858.28 3.92E+05 8909.51 5.59E+02 Cd 111 1.05E+00 0.01 2.12E+00 0.02 5.20E+00 0.05 1.70E-01 Co 59 8.75E+01 1.48 1.50E+02 2.54 6.53E+02 11.06 1.80E-01 Cr 52 2.75E+01 0.53 2.81E+01 0.54 4.43E+01 0.85 7.31E+00 Cu 63 9.82E+01 1.56 1.57E+02 2.49 5.16E+02 8.19 1.47E+00 Fe 57 2.73E+04 478.40 4.54E+04 797.01 2.27E+05 3988.57 1.72E+02 K 39 4.27E+03 109.53 6.39E+03 163.74 1.40E+04 359.35 1.50E+02 Mg 24 5.06E+04 2108.62 8.88E+04 3700.95 3.93E+05 16393.91 6.93E+00 Mn 55 2.63E+03 47.73 5.61E+03 101.96 2.50E+04 454.78 1.85E+00 Na 23 1.09E+04 474.40 1.81E+04 786.91 6.74E+04 2932.45 2.75E+01 Ni 60 6.11E+01 1.02 1.08E+02 1.79 4.55E+02 7.58 8.60E-01 Pb 208 1.01E+00 0.01 1.33E+00 0.01 1.58E+00 0.01 1.40E-01 Sb 121 2.62E-01 0.002 1.87E-01 0.002 1.33E-01 0.001 7.00E-02 Se 82 6.84E-01 0.01 2.03E+00 0.02 7.04E+00 0.08 8.06E+00 Sr 88 2.39E+02 2.717 4.43E+02 5.04 1.32E+03 15.06 8.00E-02 Tl 205 1.71E-01 0.001 5.80E-01 0.003 1.99E-01 0.001 9.00E-02 V 51 1.36E+01 0.27 1.33E+01 0.26 2.18E+01 0.43 6.92E+00 Zn 66 6.78E+02 10.27 1.63E+03 24.68 2.11E+03 31.93 6.77E+00 MDL: minimum detection limit; BDL: below detection limit; ppb: parts per billion All relative standard deviation values were ≤ 5 %, except for Ag, Cd, Pb, and Sb which were between 9-24% Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  94   Role of pH in the antibacterial activity of KC       Previously it was demonstrated that the broad-spectrum active component(s) of KC can be extracted with water. However, leachates prepared from KC were frequently at a pH below 4.5. While it was also shown that low pH is not solely responsible for the inhibitory activity of the KC leachates, to investigate if it is required for activity, from an original KC aqueous leachate, L50 (pH 3.8), a sample of the leachate with pH altered to 7.0 was prepared. Comparative viability assays against E. coli MG1655 revealed that the activity of KC leachates against bacteria is affected by raising its pH. Fig. 4.4 shows that increasing the pH of leachate to neutral rendered it ineffective. However, although the inhibitory activity of KC appears pH-dependent, low pH was not solely responsible for activity against E. coli. Incubation of E. coli in low-pH buffer (pH 3.8) resulted in a 3 log10 reduction within 24 h incubation.    E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524012345678dH2O ( 6.8)KC-L ( 3.8)KC-L ( 7.0)Buffer ( 3.8)Buffer ( 7.0)^ ^ Figure 4.4 Effect of pH on the antibacterial activity of KC-L (L50) From L50 (pH 3.8), a sample of leachate with pH altered to 7.0 was prepared. Viability of E. coli MG1655 treated with these leachates or 100 mM phosphate buffer with the equal pH have been compared.  The dotted line at log10=1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. (pH values in parentheses)  Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC  95   pH adjustment experiments and precipitate formation       Antibacterial activity of KC-L is pH-dependent. Adjusting the pH of leachate to a neutral pH rendered the leachate inactive and formed dark yellow precipitates (Fig. 4.5A, B). The precipitates were collected and resuspended in sterile dH2O. Performing comparative antibacterial assays against E. coli MG1655 revealed that collected supernatants did not exhibit antibacterial activity. As it could be due to their pHs (7.0 and 6.85 for L50 and L100, respectively), readjusting the pH to that of original leachates did not rescue the activity (Fig. 4.6A, 4.7A). In contrast, resuspended precipitate fractions with original pH about 6.1 reduced the CFUs around 1 log10 within 24 h of treatment, while after pH readjustment to the original leachate pH, they exhibited potent antibacterial activity suggesting that precipitate fractions harbor most of the activity (Fig. 4.6B, 4.7B). These results were used as guidance to analyze the precipitates for identifying the active principal components of KC-L activity.            Figure 4.5 KC-L (L50, L100) with the precipitates formed after pH-adjustment to 7.0 (A), collected precipitates after centrifugation (B)   Collected precipitates A B L50 L100 96 E . c o li  M G 1 6 5 5Log10 CFU/mL0 524 0 524 0 524 0 524 0 524012345678dH2OL50L50 supernatant ( 7.0)L50 supernatant ( 3.8)Buffer 3.8^Log10 CFU/mL0 524 0 524 0 524 0 524 0 524012345678dH2OL50 ( 3.8)L50 precipitate ( 6.1)L50- precipitate ( 3.8)Buffer 3.8^ ^Figure 4.6 Antibacterial activity of supernatant (A), and resuspended precipitate (B) compared to the original KC-L (L50) against E. coli MG1655 The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. ^ indicates that no viable cell could be recovered at that time point. Error bars represent the standard error (SE) of the mean of at least three independent replicates. (pH values in parentheses). A B 97 E . c o li  M G 1 6 5 5Log10 CFU/mL0 524 0 524 0 524 0 524 0 524012345678dH2OL100 ( 3.65)L100 supernatant ( 6.85)L100 supernatant ( 3.68)Buffer 3.8^ ^Log10 CFU/mL0 524 0 524 0 524 0 524 0 524012345678dH2OL100 ( 3.65)L100 precipitate ( 6.11)L100 precipitate ( 3.66)Buffer 3.8^ ^ ^Figure 4.7 Antibacterial activity of supernatant (A) and resuspended precipitate (B) compared to the original KC-L (L100) against E. coli MG1655  The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. ^ indicates that no viable cell could be recovered at that time point.  Error bars represent the standard error (SE) of the mean of at least three independent replicates. (pH values in parentheses).  A B Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 98 Elemental analyses of precipitates by ICP-MS and ICP-OES      Elemental analysis of original leachates and collected supernatants revealed that the precipitate fractions contained most of the Al, Be, Co, Cu, Fe, Ni, and Zn (Table 4.3). In addition, to investigate the presence of sulfur in the precipitates, ICP-OES analysis was performed. The results showed that precipitate fractions of L50 and L100 contained 0.88% and 0.95% of leachates’ sulfur, respectively. Table 4.3 ICP-MS analysis of KC-L (L50, L100), and supernatants after collecting the precipitates Amounts are reported in µg/L.     Element L50 L100 amounts in µg/L Initial-L Supernatant Difference % in precipitate Initial-L Supernatant Difference % in PrecipitateAl 27 1.03E+04 6.60E+01 1.02E+04 99.36 2.45E+04 6.55E+01 2.44E+04 99.73As 75 7.65E-01 1.86E+00 na  - 1.14E+00 7.22E-01 4.20E-01 36.80Ba 138 1.61E+01 1.47E+01 1.43E+00 8.87 1.90E+01 1.40E+01 5.00E+00 26.30Be 9 6.53E+00 4.07E-02 6.49E+00 99.38 1.26E+01 2.27E-02 1.26E+01 99.82Cd 111 1.68E+00 4.58E-01 1.22E+00 72.67 1.57E+00 2.59E-01 1.31E+00 83.46Co 59 9.83E+01 3.59E+01 6.25E+01 63.52 2.06E+02 5.23E+01 1.54E+02 74.62Cr 52 9.40E-02 1.56E-01 na  - 1.78E+00 1.82E-02 1.76E+00 98.98Cu 63 1.83E+02 2.20E+01 1.61E+02 88.00 2.72E+02 1.36E+01 2.59E+02 95.01Fe 57 3.76E+04 7.40E+02 3.68E+04 98.03 5.90E+04 1.19E+03 5.78E+04 97.98Mg 24 6.41E+04 5.59E+04 8.16E+03 12.74 1.30E+05 1.17E+05 1.35E+04 10.37Mn 55 4.21E+03 3.32E+03 8.87E+02 21.08 8.51E+03 6.38E+03 2.13E+03 25.08Ni 60 9.82E+01 4.73E+01 5.09E+01 51.82 2.21E+02 7.79E+01 1.43E+02 64.74Pb 208 8.85E-01 3.48E-01 5.37E-01 60.65 7.11E-01 4.41E-01 2.70E-01 37.99Sb 121 9.07E-02 2.46E-01 na  - 6.28E-02 6.58E-02 na  -Se 82 1.14E+00 2.08E+00 na  - 1.38E+00 1.44E+00 na  -Sr 88 4.04E+02 3.67E+02 3.75E+01 9.29 7.38E+02 6.24E+02 1.14E+02 15.48Tl 205 7.25E-02 2.28E-01 na  - 1.60E-01 1.36E-01 2.41E-02 15.05V 51 2.52E-01 1.20E+00 na  - 1.71E-01 3.79E-01 na  -Zn 66 6.63E+02 4.09E+01 6.22E+02 93.82 7.90E+02 3.68E+01 7.53E+02 95.34To narrow down the number of elements for preparation of the artificial metal ion mixtures, any element with more than 50%  of initial concentrations in precipitates (both L50 and L100) was chosen among which Be and Cd were omitted due to the high toxicity and low detected amount, respectively. Six elements shaded in blue were used for making the artificial metal ion mixture (MIM).  na: not applicable. Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 99 Antibacterial activity of single metal ion (Fe2+, Fe3+, Al3+) solutions and metal ion mixture (MIM) on the viability and growth of bacteria      Using the ICP-MS elemental analysis presented in Table 4.2, three single metal chloride solutions of Fe2+, Fe3+, and Al3+ at the same concentration and pH as KC-L100 were prepared. In addition, based on the pH studies and the chemical analysis of precipitates (Table 4.3), a metal-ion mixture (MIM) of six metal ions, Al3+, Co2+, Cu2+, Fe2+, Ni2+, and Zn2+ was prepared from their chloride salts. The antibacterial activity of single metal solutions and MIM was tested on the viability of E. coli MG1655, S. aureus RN4220, and P. aeruginosa PAO1 compared to the original KC-leachate (L100). As shown in Fig. 4.8A, L100 reduced E. coli MG1655 viability below the detection limit within 5 h of incubation, while FeCl2 and MIM exhibited the same inhibitory effect within 24 h. Both FeCl3 and AlCl3 showed about 5 log10 decline in the number of viable E. coli cells after 24 h. For S. aureus RN4220, L100, FeCl2, AlCl3, and MIM lowered the viability below the detection limit within 24 h of treatment, whereas FeCl3 caused ~4 log10 reduction in CFUs (Fig. 4.8B). In addition, P. aeruginosa PAO1 was most sensitive to metal solutions. While L100 and MIM completely eliminated P. aeruginosa PAO1 within 5 h, the same effect took 24 h of incubation for FeCl2, FeCl3, and AlCl3 solutions (Fig. 4.8C).  E . c o li  M G 1 6 5 5T im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524 0 524012345678^^AlCl3MIML100dH2OFeCl3FeCl2^^A 100 S .a u re u s  R N 4 2 2 0T im e (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524 0 524012345678dH2OL100FeCl2FeCl3^ ^AlCl3MIM^ ^P . a e ru g in o s a  P A O 1T im e (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524 0 524012345678dH2OL100FeCl2FeCl3^ ^ ^AlCl3MIM^ ^ ^ ^Figure 4.8 Effect of KC-L (L100) compared to single metal ion solutions (FeCl2, FeCl3, and AlCl3) and metal ion mixture (MIM) on the viability of E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. C B Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 101      To investigate the comparative antibacterial action of KC-L with metal ion solutions on the growth of bacteria, a defined minimal medium (MBMM) was used. As shown in Fig. 4.9, FeCl2 and MIM exhibited potent antibacterial activity against E. coli MG1655 and P. aeruginosa PAO1 with complete killing effect within 24 h of treatment, while AlCl3 caused about 5 log10 reduction or complete elimination of CFUs respectively. Moreover, treatment of S. aureus RN4220 caused more than 2 log10 decline in CFUs in 24 h, MIM eliminated the viability below the detection limit in the same period. No growth inhibitory activity was observed for FeCl3 against these bacteria. T im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524 0 524 0 5240123456789MBMMMBMM+ dH2OMBMM+ L100MBMM+ FeCl2MBMM+ FeCl3E . c o li  M G 1 6 5 5^^ ^MBMM+ AlCl3MBMM+ MIM^^A 102  S .a u re u s  R N 4 2 2 0Log10 CFU/mL0 524 0 524 0 524 0 524 0 524 0 524 0 5240123456789MBMMMBMM+ dH2OMBMM+ L100MBMM+ FeCl2MBMM+ FeCl3T im e  (h )MBMM+ AlCl3MBMM+ MIM^T im e  (h )Log10 CFU/mL0 524 0 524 0 524 0 524 0 524 0 524 0 5240123456789MBMMMBMM+ dH2OMBMM+ L100MBMM+ FeCl2MBMM+ FeCl3^ ^ ^P . a e ru g in o s a  P A O 1MBMM+ AlCl3MBMM+ MIM^^Figure 4.9 Antibacterial activity of KC leachate (L100) compared to single metal ion solutions (FeCl2, FeCl3, and AlCl3) and metal ion mixture (MIM) on the growth E. coli MG1655 (A), S. aureus RN4220 (B), and P. aeruginosa PAO1 (C) in a defined-minimal medium (MBMM) The dotted line at log10 =1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates.   B C Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 103 MIC and MBC of metal ion solutions      Minimum inhibitory and bactericidal concentrations of metal ion solutions were determined for three bacterial strains using MBMM broth (Table 4.4).  In addition, to investigate the impact of media components on the metal ion toxicity, MIC and MBC values were compared in rich and minimal media (Table 4.5).  Table 4.4 MIC and MBC values of single metal ion solutions for three bacterial strains growing in MBMM broth  E. coli MG1655 P.aeruginosa PAO1 S. aureus RN4220Metal ion MIC MBC MIC MBC MIC MBCAl3+ 0.30 1.09 0.73 1.20 1.20 3.13 Fe2+ 0.25 0.73 0.36 0.89 0.73 1.25 Fe3+ 0.49 2.40 0.89 2.40 1.87 3.75 Co2+ 0.16 0.59 0.22 0.64 0.11 0.25 Cu2+ 0.06 0.08 0.45 0.64 0.25 0.64 Ni2+ 0.08 0.46 0.45 1.38 0.17 1.73 Zn2+ 0.17 0.23 0.64 1.03 0.17 0.45 The assays were performed in minimal MBMM broth with the described supplementations. Recorded amounts are the mean of three independent experiments. (amount recorded in mM) Table 4.5 MIC and MBC values of metal ion solutions for E. coli MG1655 growing in four different broth media LB-Miller LB-Lennox LB without salt MBMM Metal ion MIC MBC MIC MBC MIC MBC MIC MBCAl3+ 12.50 25.00 12.50 12.50 6.25 6.25 0.30 1.09 Fe2+ 6.25* 12.50 6.25* 12.50 6.25* 6.25 0.25 0.73 Fe3+ 6.25* 12.50 6.25* 6.25 6.25* 6.25 0.49 2.40 Co2+ 2.03 2.81 2.50 2.50 1.02 1.88 0.16 0.59 Cu2+ 4.06 5.63 2.5-5 5.00 0.84 4.38 0.06 0.08 Ni2+ 4.06 6.25 5.00 6.25 3.28 5.63 0.08 0.46 Zn2+ 2.03 2.81 2.50 2.50 2.03 2.81 0.17 0.23 The assays were performed in three types of LB broth and also minimal MBMM supplemented with 0.01% (wt/vol) NZ-amine. Recorded amounts are the mean of three independent experiments. Amounts recorded  in mM. * Metal precipitate formation observed for Fe2+ and Fe3+ solutions, added to LB broth media, might affect the MIC values. Thisreflects in part the effect of media components on the metal solubility and bioavailability.Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 104 Discussion      Certain metal ions are essential for the life of all living organisms; while, all metals exert toxicity at elevated concentrations (Gadd 1992; Weiss and Carver 2018; Chandrangsu et al. 2017). These toxic metals interact with essential cellular components through ionic and covalent binding and can disrupt a variety of cellular functions, impair cell membranes integrity, alter enzyme specificity, and damage the structure of DNA (Bruins et al. 2000). Despite the activation of a range of adaptive responses to excess metals for maintaining hemostasis (i.e. expression of efflux systems, metal sequestration and storage, or abundance of metal-binding metabolites), excess metals, eventually cease growth and kill bacteria, often by virtue of mismetallation of metalloproteins and ultimate metal intoxication (Nies 2003; Chandrangsu et al. 2017; Lemire et al. 2013).      Due to their potent antimicrobial properties, specific metals have been exploited through history as biocides or antimicrobial agents, despite doubts about their host toxicity (Turner 2017; Hobman and Crossman 2015). The application of metals in medicine was common until the discovery of antibiotics. Nevertheless, at the beginning of the 21st century, the rapid emergence of antimicrobial resistance along with a lack of new antibiotic drugs brought a revival of interest in the utilization of metal-based antimicrobial agents, owing to their potency and less possibility of the evolution of resistance (Turner 2017). Whilst, traditional antibiotics mostly follow the “bullet-target concept”, by targeting specific biochemical processes or other metabolic key enzymes, which in turn provide ease for resistance development; metal ions alternatively revealed to inhibit multiple cellular processes simultaneously, causing pleiotropic effects on microbial cells (Nies 1999; Harrison et al. 2007; Gold et al. 2018).       The studies presented here demonstrated that the acidic nature of KC leachates is required for their activity, which suggests that certain acid-soluble metal ions may contribute to KC antibacterial activity. Furthermore, elemental analyses of KC aqueous leachates revealed that this clay releases large amounts of Al and exchangeable transition metal ions, particularly Fe, Cu, Mn, and Zn at µM to mM concentrations (Table 4.2). In addition, the enhanced survival of E. coli in KC-L treated with metal Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 105 chelators, EDTA, a broad-spectrum cation chelator, and BPY and DFO, two known Fe chelators suggests that chelated cations are major contributors in the antibacterial activity of leachates (Table B.1) (Keberle 1964; Flora and Pachauri 2010). The reported hierarchy of EDTA relative binding affinities for metal ions is as follows: Cr2+> Fe3+> Cu2+> Pb2+> Zn2+> Cd2+> Co2+> Al3+> Fe2+> Mn2+> Ca2+> Mg2+ (Smith 2013). While both DFO and BPY have been reported to form stable complexes with Co2+, Cu2+, Fe2+, Ni2+, and Zn2+  with different pKs values, DFO is able to chelate Fe3+ , Al3+, and Cr3+ (Makrlik and Van̆ura 1992; Kontoghiorghes 1995; and Keberle 1964; Flora and Pachauri 2010). DFO forms 1:1complexes with Fe whereas three molecules of BPY are necessary to coordinate with one Fe ion (Elandallousi et al. 2003; Gaeta and Hider 2005). These may partly explain the higher potency of DFO in eliminating the activity of treated KC-L compared to that of the BPY. These observations provide more insight into the mechanism of leachate action, and are consistent with the role of acid-soluble metal ions in the antibacterial activity of KC.       Previous studies suggested a role for exchangeable cations in the bactericidal activity of CsAg02 and OMT (Williams et al. 2008; Morrison et al. 2014, 2016). Otto et al. (2010) reported that aqueous extracts of two different natural antibacterial clay mixtures, BY07 and CB07, maintained their antibacterial activity, demonstrating that the in vitro antibacterial activity of the natural clay may depend on chemical desorption of specific metal ions from the surface of the clay particles. Otto and Haydel (2013a) demonstrated that mineral clays exhibiting cidal activity contained higher concentrations of chemically accessible metal ions than non-antibacterial samples and further speciation modeling and cation supplementation indicated the specific roles of Fe2+, Cu2+, and Zn2+ in the antibacterial activity of certain clay minerals.      Essential micronutrients such as Co2+, Ni2+, and Zn2+ are required at nM concentrations for bacteria, while they exert toxicity at µM or mM amounts (Nies 1992). It was demonstrated that KC aqueous leachates contain large amounts of Al and exchangeable metal ions at µM to mM concentrations (Table 4.2). Due to the multiplicity and diversity of roles for different metals in bacteria, the simultaneous effect Chapter 4: Roles of pH and Metal Ions in the Antibacterial Activity of KC 106 of different metals at toxic levels perturbs the delicate balance of metal ion homeostasis, and can influence almost every aspect of cell metabolism and growth to varying degrees (Gadd 1992; Chandrangsu et al 2017). In such conditions, lack of allosteric inhibition for most of high-affinity metal uptake systems can exacerbate the metal toxicity; moreover, some metal ions such as Co2+, Cd2+, and Zn2+ inhibit key cellular processes such as the electron transport chain extracellularly (Chandrangsu et al. 2017).       Furthermore, to investigate the key principal components of KC-L, an artificial metal ion mixture (MIM) with a limited number of metals, simulating the L100 concentrations was prepared. MIM exhibited potent inhibitory action on the viability and growth of bacteria. While in viability assays the antibacterial activity of Fe3+ solution was observed, in contrast, no inhibitory effect on growing bacteria in MBMM was detected. Sun et al. (2011) showed that the antibacterial effects of buffered Fe3+ solutions were weaker than those of Fe3+ aqueous solutions, thus higher concentrations of FeCl3 are required in buffered solutions to achieve comparable antibacterial efficiencies.        MIC values are applied to determine the susceptibilities of bacteria to antibacterial agents, and also to evaluate the activity of new antimicrobial agents. In addition, MBCs provide a general indication of bactericidal activity, while combination of MIC and MBC together with time–kill curves yield a more meaningful measurement (Wiegand et al. 2008). In general, MIC and MBC values were greater in rich media than in minimal MBMM, which may explain why those media were not suitable for investigating growth inhibition assays of KC-L or metal studies. The required higher concentrations of metal ions in rich media may reflect the lower bioavailability of metals due to chelating effects of organic matter as well as buffering agent present in the rich growth media (Harrison et al. 2005; Rathnayake et al. 2013). Collectively, these results provide a better understanding of the role of metal ions in the antibacterial activity of KC as well as metal-bearing clay minerals, which is in turn vital to standardizing KC, making effective antibacterial clays, and also for future design of MBA agents. 107  Elucidating the Mode(s) of Action of KC Leachates Introduction      Reactive oxygen species (ROS) including superoxide anions (O2•−), hydroxyl radicals (OH•), and hydrogen peroxide (H2O2) are inescapable by-products of normal aerobic bacterial metabolism (Imlay 2009). Due to their highly reactive feature, they exert toxic effects on bacteria and damage essential macromolecules, alter their function, and leading to cell death (Imlay 2013; Mishra and Imlay 2012; Ezraty et al. 2017). Virtually, all aerobic microorganisms have evolved complex repair and defense mechanisms by encoding multigene responses to mitigate the deleterious effects of ROS (Farr and Kogoma 1991). Efficient enzyme machinery scavenge and decrease ROS and repair the oxidative damage to biomolecules (Imlay 2009; Lushchak 2001). Oxidative stress has been functionally defined as an excess of prooxidants, beyond the capacity of cellular defenses (Farr and Kogoma 1991).      In aerobic environments, ROS can form endogenously via the reaction of O2 and univalent electron donors such as metal centers (Ezraty et al. 2017). Mediated by the Fenton reaction, OH• radicals are generated by the reduction of hydrogen peroxide in the presence of redox metal ions (Fe2+, Fe3+, or Cu+), which damage bacterial DNA, RNA, proteins, and lipids (Mishra and Imlay 2012; Imlay et al. 1988). Oxidative damage can result in covalent modifications that destabilize proteins (Ezraty et al. 2017). An excess of certain reduced metal ions, especially Fe2+ and Cu2+, catalyze the formation of ROS via Fenton chemistry (Valco et al. 2005; Lemire et al. 2013). Thus, toxicity associated with these metals might be due, at least in part, to ROS-mediated cellular damage (Lemire et al. 2013). Additionally, superoxide favors the Fenton reaction by releasing iron from iron-containing proteins (Imlay 2013).        Bacteria have evolved defense strategies and repair programs to protect against the damage caused by oxidative stress (Imlay 2013). Excess amounts of free intracellular iron and deregulation of iron homeostasis can lead to oxidative stress (Touati 2000). This may be induced in bacteria by exogenous biocidal compounds or inorganic nanoparticles (Cohn et al. 2006b; Moos and Slaveykova 2014). Pyrite Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 108 (FeS2) as the most abundant metal sulphide on the Earth can spontaneously generate the ROS, hydrogen peroxide, and hydroxyl radicals, when exposed to water (Cohn et al. 2006a; Cohn et al. 2010; Javadi Nooshabadi and Rao 2014). Fe-rich clay minerals such as hematite and magnetite and expandable 2:1 phyllosilicates induce oxidative stress via lipid peroxidation (Watts et al. 1999; Kibanova et al. 2009). Furthermore, exogenous hydrogen peroxide-induced oxidative stress damages bacterial phospholipid membranes via lipid peroxidation (Lemire et al. 2013; Imlay 2003). Recent studies, reporting the accumulation of iron in bacteria treated with clay minerals, suggest a role for ROS in their lethal activity (Morrison et al. 2014, 2016). Collectively, the toxicity of exchangeable metal ions inherent in KC might be due, at least in part, to ROS-mediated cellular damage.      The bacterial cell membrane (CM) is responsible for many key functions as it maintains selective permeability for cellular homeostasis, osmoregulation, and metabolic energy transduction (Zhang and Rock 2008; Silhavy et al. 2010). The CM contains about one-third of all bacterial proteins and is the site for pivotal cellular processes including active transport, bacterial respiration, establishment of the proton motive force, biosynthesis, and ATP generation for which membrane integrity is fundamental. Its disturbance can directly or indirectly cause metabolic dysfunction and cell death (Hurdle et al. 2011; Strahl and Errington 2017). Antimicrobial peptides and several bioactive compounds that affect the membrane validate its importance as an antibacterial target site (Hurdle et al. 2011; Fjell et al. 2012; Epand et al. 2016).       Bacterial membranes contain polymers with highly electronegative chemical groups, which serve as sites of adsorption for metal cations and cationic compounds (Lemire et al. 2013). Due to the ability of CM to coordinate metals, it has been suggested that it is the target at which some metals exert bactericidal toxicity (Vaara 1992; Nikaido and Vaara 1985). The outer membrane (OM) permeabilizing effect of high concentrations of Na+ and divalent cations, especially Ca2+ and Mg2+, has been reported (Homma and Nakae 1982; Brass 1986; Vaara 1992). Moreover, electron microscopy (EM) studies have shown that the integrity of the bacterial CM is compromised by exposure to toxic doses of Ag and Al (Feng et al. 2000; Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 109 Jung et al. 2008; Yaganza et al. 2004). Lipid peroxidation has been associated with Cu2+ and Cd2+ toxicity in bacteria and yeast (Hong et al. 2012; Howlett and Avery 1997), while exogenous hydrogen peroxide-induced oxidative stress can damage bacterial phospholipid membranes through lipid peroxidation (Imlay 2003, 2013). Some effects of this process include increased membrane rigidity, distorted permeability, and altered activity of membrane-bound enzymes and receptors, all of which could contribute to cell lysis and death (Imlay 2013). Some studies have reported cell membrane damage and permeabilization induced in bacteria following exposure to mineral clays and modified clays (Otto et al. 2010; Kibanova et al. 2009; Su et al. 2009). This chapter investigates the second research aim by elucidating the ROS generation and oxidative stress formation in bacteria treated with KC leachate. In addition, this chapter aims to elucidate any disruption in bacterial cell membrane integrity induced upon treatment with KC-L to aid in understanding the role of the bacterial membrane as a possible target in KC toxicity.   Materials and methods Bacterial strains and growth conditions E. coli MG1655 and S. aureus RN4220 were used as representative Gram-negative and Gram-positiveorganisms, respectively. E. coli MG1655 was grown in LB broth or on LB agar, while S. aureus RN4220 was grown in TSB or on TSA as described in section 3.2.1. To investigate the role of oxidative stress in the activity of KC-L, a collection of 10 mutant strains of E. coli MG1655, as described in Table 5.1, were studied.  110 Table 5.1 Oxidative-stress and DNA-damage related mutant strains of E. coli MG1655 used in this study No.  Name Mutant Genotype/characteristics Reference 1 JI360 katE As MG1655 plus katE12::Tn10 Seaver and Imlay 2001 2 JI361 katG As MG1655 plus katG17::Tn10 Seaver and Imlay 2001 3 JI367 katE katG As MG1655 plus ΔkatG katE12::Tn10 Park and Imlay 2003 4 JI370 ahpCF As MG1655 plus ΔahpCF' kan::'ahpF Seaver and Imlay 2001 5 JI372 katE ahpCF As MG1655 ΔahpF::kan Δ(katE12::Tn10)1 Mancini and Imlay 2015 6 JI374 katG ahpCF As JI364 plus ΔahpCFΔkan::'ahpF Seaver and Imlay 2001 7 AL427 Hpx- or (katG katE ahpCF) As MG1655 plus Δ(ahpCF::cat)1 Δ(katG17::Tn10)1 Δ(katE12::Tn10)1 Liu and Imlay 2013 8 KI232 sodA sodB As MG1655 plus (sodB-kan)1 Δ(sodA::cat)1  Liu and Imlay 2013 9 AL486 recA As MG1655 plus Δ(recA774::kan)1  Liu and Imlay 2013 10 AL410 polA1 As MG1655 plus polA1~zih-102::Tn10  Liu and Imlay 2013 All the strains are congenic derivatives of E. coli MG1655 (WT). Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 111 Antibacterial assays Antibacterial activity of KC leachate under aerobic vs. anoxic condition      The most direct way to assess the role of ROS in KC toxicity would be to examine the antibacterial activity of KC under anaerobic conditions, when ROS cannot be formed. The antibacterial activity of KC under aerobic and anaerobic conditions against E. coli as a facultative anaerobic bacterium was compared as described by Liu and Imlay and Keren et al. (2013) with some modifications. Briefly, mid exponential growth phase culture of E. coli MG1655 was prepared as described in section 3.2.6.1. As E. coli MG1655 exhibits histidine auxotrophy under anaerobic condition, 0.5 mM histidine (Sigma) was added to the MES-buffered minimal medium MBMM broth, supplemented with 0.1% NZ-amine (Xu and Imlay 2012). To minimize the difference between oxic and anoxic cultures, oxic cultures were supplemented with histidine as well (Jang and Imlay 2010). For anoxic experiments, the tubes were perfused with N2 gas for 5 min prior to inoculating them with a washed pellet of mid-exponential phase cultures of bacteria. The treatments with dH2O as control or KC-L (L100) were transferred to an anaerobic jar (Oxoid) containing an anaerobic GasPak (Oxoid) and incubated at 37˚C for 24 h with gentle shaking at 150 rpm. An OxoidTM resazurin anaerobic indicator (Thermo Fisher) was used as the quality control for the anaerobic condition. CFUs were determined by 10-fold serial dilution plating of aliquots removed at the start of experiments and after 24 h of treatments. Catalase supplementation of growth media      To investigate whether supplementation of growth media with an oxidative stress protectant affects the sensitivity of E. coli cells to KC-L or delays killing, catalase was used. 10 mg catalase (Bovine liver catalase, ~2,390 U/mg solid, Sigma) was dissolved in sterile dH2O and then filtered through a Millex-HV 0.45 µm filter. Washed pellet of mid-exponentially growing cells of E. coli MG1655 was prepared as described in section 3.2.6.1 and resuspended in MBMM defined medium supplemented with 0.02% NZ amine at a concentration of ~107 CFU mL-1. Bacterial suspensions were treated with either KC-L (L100) Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  112  or dH2O. To investigate the effect of catalase, a freshly prepared catalase solution was added to a series of treatment tubes and controls at 25 µL mL-1. The tubes were incubated at 37˚C with shaking at 200 rpm. CFUs were determined by 10-fold serial dilution plating of aliquots removed at the start of experiment and after 2.5, 5, 8, and 24 h of treatments. In order to scavenge H2O2, all the 10-fold serial dilution microtubes containing 900 µL NS solution and LB agar plates were also supplemented with 50 and 55 µL of catalase suspension respectively, prior to use.   Antibacterial activity of KC leachate against oxidative stress related bacterial mutants        To investigate whether ROS generation is a significant component of the antibacterial activity of KC leachate, the following experiment was carried out. The effects of mutations in oxidative stress defense genes, known to reduce the levels of ROS in bacteria (such as katE and katG (catalase), sodA, and sodB (superoxide dismutase), and ahpCF (alkyl hydroperoxide reductase)) on the sensitivity of E. coli cells to KC-L were studied (Table 5.1). The response of an Hpx- mutant that lacks catalase and peroxidase activities to KC-L was also examined. In addition, since the most consequential impacts of oxidative stress are DNA damage and mutagenesis (Imlay 2013), two mutant strains deficient in recombination or excision repair (recA and polA1) were studied. To prevent the photochemical generation of H2O2, LB plates were prepared freshly or one day previously and stored in the dark.         The present study was carried out as described in section 4.2.7. Briefly, mid-exponential phase grown E. coli MG1655 (wild-type parental strain) as well as mutant strains were prepared in LB broth. Cells were then harvested by centrifugation, rinsed once in sterile PBS (pH 7.4), and resuspended in MBMM broth supplemented with 0.02% NZ amine at an initial cell concentration of ~107 CFU mL-1 and then treated with KC-L (L100) or dH2O in a (1:1) ratio. Suspensions were then incubated at 37˚C with shaking at 200 rpm and CFUs were determined by 10-fold serial dilutions and duplicate plating of aliquots removed at the start of experiments and after 2.5, 5, 8, and 24 h of treatment.    Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 113 Transmission electron microscopy (TEM) studies      To investigate morphological changes induced in bacteria following exposure to KC-L, treated E. coli MG1655 and S. aureus RN4220 were analysed by TEM at three time points of a viability assay. Briefly, washed pellets of mid-exponential phase growth of E. coli MG1655 and S. aureus RN4220 were prepared as described in section 3.2.6.1.  Bacterial cells were suspended in KC aqueous leachate (L500) (pH 4.4), in dH2O with neutral pH, and in dH2O at the same low-pH as L500 (4.4) in a viability assay as previously described in section 3.2.6.1. At three different time points (0, 5, and 24 h) of the assay, aliquots of bacterial suspensions were deposited on TEM copper grids (Sigma) covered with carbon film, stained negatively with 2% aqueous phosphotungstic acid (PTA), and then were observed with a  Hitachi H7600 TEM. A minimum of 50 cells was observed from each of three treatments.  Outer membrane permeability assay      Outer membrane (OM) permeabilization by KC leachate was determined using the hydrophobic fluorophore N-phenyl-1-naphthylamine (NPN) uptake assay as described by Helander and Mattila-Sandholm (2000), Chusri et al. (2009), and Gerits et al. (2016). Briefly, NPN (Sigma) was dissolved in acetone at a concentration of 500 mM and then a 40 µM working solution was prepared by diluting the stock solution in sterile dH2O and kept in the dark at -20˚C. Mid exponential growth of E. coli MG1655 was prepared in LB broth as described in section 3.2.6.1. The cells were collected by centrifugation and washed once with sterile normal saline (0.85% wt/vol) and resuspended in sterile 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 7.24) to a final concentration about 107 CFU mL-1. Then, 80 μL aliquots of bacterial suspensions were transferred to a 96-well optical-bottom black microtiter plate (Thermo Fisher Scientific) and treated with 85 μL of either KC-L (L100), EDTA solutions (0.25 mM and 1 mM) as positive controls, or HEPES buffer (5 mM) as a negative control and then NPN was added to the cell suspensions to a final concentration of 10 μM. Uptake assay was performed in a temperature-controlled microplate reader (on a Varioscan Flash Spectral Scanning Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 114 Multimode Microplate Reader (Thermo Scientific) at 30˚C. Fluorescence intensities at an excitation and emission wavelength of 355 and 460 nm, respectively, as well as optical densities (OD600) were measured within 3 min and continuously recorded every 10 minutes for 3.5 h. Each assay was performed at least three times. Fluorescence values were corrected for background fluorescence by subtracting the values of the untreated control cultures stained with NPN probe and then values were divided by corresponding OD600 values to normalize upon cell densities. The alteration in OM permeability was also evaluated, in the same approach, upon treatment with single metal ion solutions (AlCl3, FeCl2, FeCl3), and MIM, prepared as described in section 4.2.6. Each assay was performed three times. BacLight Live/Dead assay and flow cytometry      To assess the membrane integrity of E. coli following treatment with KC leachates, the BacLight LIVE/DEAD membrane permeability kit (Invitrogen) was used as described by Berney et al. (2007) following the manufacturer’s guidelines. The kit is composed of two nucleic-acid binding fluorochromes: (a) SYTO9 that readily penetrates intact or impaired membranes, binds to nucleic acids, and inducesfluorescence emission and (b) propidium iodide (PI) which is able to cross impaired membranes. Although this kit enables differentiation between bacteria with intact and impaired CM, it has been widely used to differentiate between active and dead cells following antimicrobial treatments (Berney et al. 2007; Li et al. 2015; Bankier et al. 2018).      Briefly, a mid-exponential phase culture of E. coli MG1655 was prepared as described in section 4.2.7. The cells were harvested by centrifugation, washed once with PBS and resuspended in MBMM at an initial concentration of 108 CFU mL-1. Following exposure to the KC leachate (L100) or control conditions (dH2O or 100 mM phosphate buffer (pH 3.7), cells were incubated in 5 μM SYTO9 and 30 μM propidium iodide (PI) for 15 min in the dark and then immediately analyzed by flow cytometry. An Attune NxT, Acoustic Focusing Cytometer (Thermo Fisher) fitted with a 488 and 561 nm excitation laser was used for membrane permeability analyses. The bacteria acquisition gate was determined based on Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  115  forward scatter (FSC) and side scatter (SSC) channels to exclude background noise and debris. Green fluorescence was detected using  a 488 nm laser and 530 nm bandpass filter, while red fluorescence was detected with a 561 nm laser and 610 nm bandpass filter. For each treatment, 50,000 events were collected and analyzed in flow cytometric measurements. This experiment provides observation of the intact and permeabilized cells as well as approximate quantities and the ratio of viable and dead cells.  Transcriptional changes induced by KC leachate on an E. coli reporter library       To identify any inhibition-related induction or suppression of cellular processes, a collection of 470 reporter clones of transcriptional fusions of green fluorescent protein (GFP) (intragenic regions fused to gfpmut2 on low-copy plasmids) with different promoters in E. coli K12 MG1655 was used (Zlasver et al. 2004, 2006). Overnight growth of a collection of clones was prepared by replicating them into 96-well round-bottom culture plates containing LB broth supplemented with kanamycin (25 µg mL-1) growing for 16-18 h at 37˚C with shaking at 150 rpm. From overnight cultures, mid-exponential phase growth of clones were prepared in 96-well optical-bottom black plate (Thermo Fisher Scientific) containing MBMM medium supplemented with 0.2% NZ-amine and 25 µg mL-1 kanamycin and then treated with either KC-L (L100) or sterile ultrapure distilled water (Invitrogen) as control. The plates were covered in aluminum foil and then incubated at 37˚C with gentle shaking at 150 rpm. GFP fluorescence intensities were measured at different time points (0, 5, and 24 h) at an excitation and emission wavelength of 481 and 508 nm, respectively using a Varioscan Flash Spectral Scanning Multimode Reader (Thermo Scientific). All GFP measurements were performed as described herein. Promoter activity was determined by calculating the normalized values of GFP signals divided by the normalized corresponding OD600 for three independent replicates of treated clones with KC-L compared to the control at each time points and reported as Log2 fold-change relative to the control. The results provide some insight in the KC-related transcriptional modulations in treated clones. Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  116   Results  Role of oxidative stress  Comparative antibacterial activity of KC leachate under aerobic vs. anoxic condition      The most direct way to assess the role of ROS in KC toxicity is to examine the antibacterial activity of KC under anoxic condition, when ROS cannot be formed. The effect of KC-L (L100) under oxic and anoxic conditions against E. coli, as a facultative anaerobic bacterium, was compared. Killing by KC-L was more effective under the aerobic condition while incubation in an anoxic environment allowed 10X more bacteria to survive (Fig. 5.1).     Control (oxic)L100 (oxic)Control (anoxic)L100 (anoxic)0123456789E . c o li  M G 1 6 5 5Log10 CFU/mL Figure 5.1 Comparative antibacterial activity of KC-L (L100) against E. coli MG1655 under aerobic vs. anoxic conditions MBMM media supplemented with 0.1% NZ- amine and 0.5 mM histidine. The starting inoculums for oxic and anoxic cultures were 2.07E7+/- 16.17% and 1.78E7 +/- 3.26%, respectively. The dotted line at log10=1 of the Y axis represents the limit of detection for CFU.  The effect was not significantly different (p > 0.05).  Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 117 Effect of catalase supplementation on the sensitivity of bacteria to KC leachate           Hydrogen peroxide (H2O2) is one of the ROS molecules that its presence or generation, either extracellular or intracellular, can damage bacterial cells (Imlay 2008). H2O2 is formed through chemical processes when reduced metal ions and sulfur species are present in oxygenated aqueous environments (Imlay, 2008). As KC aqueous leachates are rich in reduced metal ions and sulfur (Table 3.4), to investigate whether H2O2 stress is part of the antibacterial activity of KC aqueous leachates, catalase therapy was applied. As Fig. 5.2 illustrates, supplementation with catalase reduced the toxicity of KC leachate (L100) against E. coli MG1655 and partially rescued the treated cells. These results together suggest that the bactericidal activity of L100 is due, at least in part, to ROS-mediated cellular damage. 0 4 8 1 2 1 6 2 0 2 40123456789T im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100WT+ CatWT+ dH2O+ CatWT+ L100+ CatE . c o li  M G 1 6 5 5Figure 5.2 Effect of catalase-supplementation on the antibacterial activity of KC-L (L100) against E. coli MG1655 (WT) Cat and WT represent catalase solution and E. coli MG1655 parental strain, respectively. The dotted line at log10=1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. An aqueous solution of catalase was added to the treatments at the start of antibacterial assay in MBMM medium. Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 118 Susceptibility of bacterial strains with mutations in oxidative-stress related functions to KC leachate       As exposure to ROS damages cellular components such as DNA, proteins, and membrane lipids, most organisms produce enzymes including catalases, peroxidases, and superoxide dismutases which react with detrimental oxidants and neutralize them prior to causing deleterious cellular damage (Ezraty et al. 2017). To study the possible role of ROS generation and oxidative stress formation upon KC aqueous leachate treatment, a collection of E. coli strains with mutations in genes related to oxidative-stress enzymatic defense functions were tested. As shown in Fig. 5.3, mutants, especially with defects in katG and katE genes were more susceptible to L100 treatment. E. coli Hpx- mutants (katG katE ahpCF) lacking catalase and peroxidase activities are unable to remove H2O2 which results in substantial damage to proteins and DNA (Liu and Imlay 2013). In addition, ahpCF and sodA sodB mutant strains that lack alkylhydroperoxide reductase and superoxide dismutase activities, respectively, exhibited lower sensitivity compared to other mutants (Fig. 5.3D, H).   0 4 8 1 2 1 6 2 0 2 40123456789k a tET im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100katEkatE+ dH2OkatE+ L1000 4 8 1 2 1 6 2 0 2 40123456789k a tGT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100katGkatG+ dH2OkatG+ L100A B 119 0 4 8 1 2 1 6 2 0 2 40123456789k a tE  k a tGT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100katE  katGkatE  katG+ dH2OkatE  katG+ L1000 4 8 1 2 1 6 2 0 2 40123456789 a h p C FT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100ahpCFahpCF+ dH2OahpCF+ L1000 4 8 1 2 1 6 2 0 2 40123456789k a tE  a h p C FT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100katE  ahpCFkatE  ahpCF+ dH2OkatE  ahpCF+ L1000 4 8 1 2 1 6 2 0 2 40123456789k a tG  a h p C FT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100katG ahpCFkatG ahpCF+ dH2OkatG ahpCF+ L100C D F E Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 120 0 4 8 1 2 1 6 2 0 2 40123456789H p x-T im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100Hpx-Hpx-+ dH2OHpx-+ L1000 4 8 1 2 1 6 2 0 2 40123456789s o d A  s o d BT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100sodA sodBsodA sodB+ dH2OsodA sodB+ L100Figure 5.3 Sensitivity of E. coli mutants with defects in oxidative-stress related functions to KC-L (L100) The dotted line at log10=1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. Antibacterial activity of L100 against mutant strains with defects in oxidative-stress related was compared to that of the parental strain, E. coli MG1655 (WT), in MBMM defined medium. Susceptibility of bacterial strains with mutations in DNA repair related functions to KC leachate        One of the most consequential impacts of oxidative stress in bacteria is mutagenesis and DNA damage for which repair and post-replication recombination is an essential back-up repair strategy is essential (Imlay 2013). Fig. 5.4 illustrates that two mutant strains lacking the recombination gene (recA) or DNA polymerase (polA1) showed hypersensitivity to KC-L, suggesting that KC also caused DNA damage in treated bacteria. G H Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 121 0 4 8 1 2 1 6 2 0 2 40123456789 r e c AT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100recArecA+ dH2OrecA+ L1000 4 8 1 2 1 6 2 0 2 40123456789p o lA 1T im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100polA1polA1+ dH2OpolA1+ L100Figure 5.4 Sensitivity of E. coli mutants with defects in DNA repair to KC-L (L100) The dotted line at log10=1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. Antibacterial activity of L100 against mutant strains with defects DNA repair functions was compared to that of the parental strain, E. coli MG1655 (WT), in MBMM defined medium. Effect of KC leachate on cell membrane integrity and permeability Ultrastructural studies of treated bacteria by TEM      TEM facilitates direct observation of the morphological and structural changes induced upon various treatments and may provide useful information on the antibacterial effects and the process of inhibition of novel antimicrobial agents (Feng et al. 2000; Hartmann et al. 2010). To investigate morphological changes induced in bacteria following exposure to KC-L (L500), treated E. coli MG1655 and S, aureus RN4220 were analysed by TEM at three time points of a viability assay. The treatment with KC leachate induced significant structural alteration. As shown in Fig. 5.5, evidence of deformations, indentations and increase in roughness of the cell surface were observed in E. coli cells treated with KC-L (C, F) but not in cells treated with neutral pH (B, E), or low-pH control (D, G). Fig. 5.6 illustrates that protoplast formation (C), cytolytic damage and release of cytoplasmic content (F) can be observed in S. aureus cells treated with KC-L. In addition, membrane disruption with release of intracellular material (5.5C, F; 5.6C, F) and A B Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 122 ghost cell (empty cell envelope structure) formations with loss of cytoplasm were also observed in both E. coli and S. aureus cells treated with KC-L (Fig. 5.7B, D). Figure 5.5 Time series TEM photographs of E. coli MG1655 cells treated with KC-L (L500) compared to controls Washed pellet of mid-exponential phase growth of E. coli was suspended in L500 (pH 4.4), in dH2O (pH 6.7), or in dH2O with the same low-pH as KC-L (4.4) in a viability assay. Bacterial suspensions were deposited on TEM copper grids at three time points (0, 5, 24 hour) and were stained negatively with 2% aqueous PTA. Disruption of cell membrane with release of intracellular material (C) and deformation and indentations of the cell surface (F) can be observed in cells treated with KC-L but not in cells incubated in dH2O with pH 6.7 (B, E) or low-pH dH2O (D, G). (scale bars 500 nm); (magnified X80,000)  A B C D E F G E. coli MG1655dH2O (4.4) KC-L (4.4) dH2O (6.7) 0 hour 24 hour 5 hour 123 Figure 5.6 Time series TEM photographs of S. aureus RN4220 cells treated with KC-L (L500) compared to controls Cell with almost completely removed cell wall, spheroplast, formation (C), cytolytic damage and release of cytoplasmic content (F) can be observed in cells treated with KC aqueous leachates. (scale bars 100 nm); (magnified X80,000)  A B C D E F G S. aureus RN4220dH2O (4.4) KC-L (4.4) dH2O (6.7) 0 hour 24 hour 5 hour 124 Figure 5.7 TEM images of morphological changes induced in E. coli MG1655 (A, B), and S. aureus RN4220 (C, D) as the results of KC-L (L500) treatments  A) Cell wall rupture and perforation in E. coli after 5 hour of exposure to KC-LB) Ghost cell formation, cell envelope indentation and deformation with loss of intracellular material in E. coli after24 hour of exposure to KC-LC) Disruption of cell envelope with release of cytoplasmic content in S. aureus and most likely protoplast formationafter 5-hour incubation in KC-LD) Ghost cell formation in S. aureus RN4220 after 24-hour incubation in KC-L(scale bars 100 nm), (magnified X80,000)B C D  A Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 125 Effect of KC leachate on the OM permeability      The cell envelope of Gram-negative bacteria is a complex multilayered structure, comprised of the OM, the inner membrane, and the periplasm (Ruiz et al. 2006). NPN is an uncharged, lipophilic, nontoxic probe which is fluorescent when bound to biological membranes (Loh et al. 1984). While an intact OM is a permeability barrier, excluding hydrophobic substances such as NPN, once damaged enables NPN to partition into the hydrophobic environment of the OM and cytoplasmic membrane and results in prominent fluorescence (Vaara 1992; Helander and Maltila-Sandholm 2000). The lipopolysaccharide (LPS) molecules released in large quantities by many of the permeabilizers binds NPN and also allows fluorescence emission (Hancock and Wong 1984; Vaara 1992). As illustrated in Fig. 5.8, the elevated fluorescence values indicate a continuous uptake of NPN as a result of permeabilization of the OM in E. coli MG1655 treated with KC-L (L100). Similarly, EDTA can chelate Ca2+ and Mg2+ inherent in the OM, break the electrostatic links between LPS molecules, and eventually disorganize and permeabilize the OM (Hancock and Wong 1984; Vaara 1992). 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0051 01 52 02 53 03 54 04 5E . c o li  M G 1 6 5 5T im e  (m in u te )NPN uptake (fluorescence unit)EDTA 0.25 mMEDTA 1 mMHEPES 5 mMKC-L ( L100)Figure 5.8 Effect of KC-L (L100) on bacterial OM permeability measured by NPN uptake assay 0.25 mM and 1 mM EDTA solutions were used as positive controls sequestering divalent cations contributing to the stability of bacterial outer membrane. HEPES buffer was used as a negative control. The first time point (0 min) shown is at ~5 min after starting the treatments. Error bars represent the standard error (SE) (n=4).   Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  126   Effect of metal ions and artificial metal ion mixture on the OM permeability      To investigate whether metal ion components of KC-L play roles in its interaction on OM integrity, the effect of single metal ion solutions as well as metal ion mixture (MIM), mimicking the concentrations found in L100 as described in chapter 4, was studied. As shown in Fig. 5.9, both AlCl3 and MIM resulted in high uptake of NPN in the first hour of treatment while FeCl3 induced weak permeabilization effect. Treatment with FeCl2 did not significantly change NPN levels.     0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0051 01 52 02 53 03 54 04 5E . c o li  M G 1 6 5 5T im e  (m in u te )NPN uptake (fluorescence unit)AlCl3FeCl3FeCl2MIMKC-L( L100)EDTA 1 mM Figure 5.9 Effect of metal ion solutions (AlCl3, FeCl2, FeCl3) and metal ion mixture (MIM) compared to that of KC-L (L100) on bacterial OM permeability measured by NPN uptake assay  A solution of 1 mM EDTA was used as a permeabilizer agent. The first time point (0 min) shown is at ~5 min after starting the treatments. Error bars represent the standard error (SE) (n=4).      Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 127 Effect of KC leachate on the CM integrity of treated cells       To complement electron microscopy observations, and also detect any disruption of bacterial cell membrane (CM) permeability following exposure to KC-L, a BacLight LIVE/DEAD viability assay combined with flow cytometry was carried out. This assay contains two nucleic acid intercalating fluorescent dyes, SYTO 9 (penetrates all membranes) and PI (only passes through permeabilized membranes) to monitor membrane permeability of cells and to provide visualization and approximate quantities of the intact and compromised cells (Berney et al. 2007). After 2 h treatment, the population of cells with permeabilized CM treated with KC-L was more abundant (22.0%) than that of dH2O or low-pH buffer controls (8.11% and 3.87%, respectively) (Fig. 5.10). 128 Figure 5.10 Flow cytometric analysis of E. coli MG1655 treated with dH2O, KC-L (L100), and low-pH buffer To evaluate the membrane integrity of E. coli following exposure to KC leachate (KC-L), the BacLight LIVE/DEAD viability kit containing two fluorescent probes, SYTO9 (penetrates intact or impaired membranes) and propidium iodide (PI, only passes through impaired and permeabilized membranes) was used following analysis by flow cytometry. The population of cells in each four categories has been reported in percentage. The population of cells for each four categories are recorded in percentage. After 2 hour treatment, the population of cells with permeabilized membranes treated with KC was more abundant than that of controls (dH2O or low-pH buffer). Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 129  Transcriptional modulations upon treatment with KC leachate      To identify any inhibition-related induction or suppression of cellular processes, a collection of reporter clones of transcriptional fusions of GFP with different promoters in E. coli MG1655 (Zlasver et al. 2004, 2006) in response to treatment with KC-L was investigated. Each of the reporter clones has a fast-folding GFP fused to a full-length copy of an E. coli promoter in a low-copy plasmid, which enables assessment of gene expression within minutes with high accuracy and reproducibility (Zhang et al. 2011). The most responsive clones have been summarized in Fig. 5.11. KC-L induced DNA damage as indicated by upregulation of recX, recN, dinB, recA, polB, uvrD, gyrB, and ybfE which are known in SOS response system and repair of damaged DNA or untargeted mutations (Onnis-Hayden et al. 2009; Tucker 2002; Khil and Camerini-Otero 2002; Lan et al. 2014). The results demonstrated the activation of oxidative stress, by induction of transcriptional regulators of oxidative stress responses (soxR, soxS, and oxyR) and triggering antioxidant defenses, detoxifications, and redox control (ahpF, katE, katG, sodB, sodC, dsbG, napF) (Gou et al. 2010; Onnis-Hayden et al. 2009). In addition, transcriptional modulation of membrane ion transporters (yfbS, ynaI, fepB), non-specific porin (ompN), and genes involved in osmoprotection (mscS, dppA) were observed upon treatment (Oniis-Hayden et al. 2009).      The results indicated that in response to the presence of metals such as Al, Zn, Mg, and Fe, transcriptional modulation happened in ais, borD, fepB, yiiP, and fur with known roles in metal transport, metal detoxification, efflux of metal ions, or iron uptake regulation (Grass et al. 2005; Brocklehurst and Morby 2000; Khil and Camerini-Otero 2002). Modulation in transcription of genes involved in antibiotic resistance (marR, mdtH, emrE, sanA), resistance to bacitracin, polymyxin, or cationic peptides (pmrD, phoB) were also demonstrated (Nishino and Yamaguchi 2001; Dual and Lister 2013). Moreover, there were some evidences of activation of other stress responses such as envelop stress (rpoE), osmotic stress (mscS, betT), acid shock (asr), phosphate deprivation (phoB), while upregulation of ybjK and ybjJ illustrated the induction of biofilm formation in response to the KC-L exposure (Seputiene et al. 2003; De Las Peñas et al. 1997; Shimada et al. 2012; Kholodkin-Gal et al. 2009). 130 Continued 131 Figure 5.11 Gene expression profiles of some of the GFP clones within 24 hour treatment with KC-L (L100) A group of clones in exponential phase growth was treated with KC-L or dH2O. Expression levels of the promoters were determined by calculating the normalized values of GFP signals divided by corresponding OD600 for treated clones with KC-L compared to the control at each time points and reported as Log2 fold-change. The first time point (0 h) shown is at ~15-20 min after starting the treatments. The fold change of gene expression is indicated by color gradient and the time course of expression changes is shown from left to right. Light to dark-red spectrum indicates up-regulation; blue colors indicate down-regulation.  Table D.1 provides further description about each clone. Dural and Lister 2013; Nishino and Yamaguchi 2001; Kolodkin-Gal et al. 2009; Onnis-Hayden et al. 2009; Khil and Camerini-Otero 20102; Lan et al. 2014; Depuydt et al. 2000; Grass et al. 2005; Brocklehurst and Morby 2000; Lee et al. 2005.  Chapter 5: Elucidating the Mode(s) of Action of KC Leachates 132 Discussion      Oxidative stress occurs when an excessive production of ROS overwhelms enzymatic and/or non-enzymatic antioxidant defenses involved in detection, detoxification, or repair of resulting damage (Imlay 2013). While studies over recent decades have pinpointed the major biomolecules which oxidants can deteriorate and bacterial defense strategies to alleviate oxidative burst, there is still lack of knowledge about the circumstances under which oxidative stress happens, partly due to the transition feature of short-lived ROS which makes their accurate detection and identification challenging (Imlay 2015, 2019; Lemire et al. 2013). Bacteria are in continuous contact with ROS generated both endogenously, as a normal product of their aerobic metabolism, and exogenously due to the environmental factors (Imlay 2008). Excessive oxidants induce alterations to the normal structure or function of bacterial macromolecules, which trigger deleterious oxidative damage, mutagenesis, and ultimately cell death (Imlay 2009). Oxidative stress can also arise via perturbation of bacterial metal homeostasis, especially Fe (Lemire et al. 2013; Chandrangsu et al. 2017). Many studies have illustrated the metal-induced oxidative stress and reported that toxic amounts of certain metals, especially Fe2+ and Cu2+, elevate intracellular ROS (Imlay et al. 1988; Warnes et al. 2012; Lemire et al. 2013). The three major mechanisms proposed for elevated ROS production during metal intoxication include: a) Fenton chemistry reactions due to certain redox-active transition metals, notably Fe, Cu, Co, Cr, and Ni; b) destruction of [4Fe-4S] clusters of proteins which could result in the release of additional Fenton-active Fe into the cytoplasm and eventually increase ROS generation; and c) generation of ROS through intermediate S radical chemistry due to thiol-mediated reduction of metal ions such as Fe3+ and Cu2+ (Lemire et al. 2013).       As any elevation in the intracellular levels of ROS, notably hydrogen peroxide (H2O2) and superoxide (O2-), can damage DNA, proteins, and lipids, bacteria have acquired defensive systems (Imlay 2009). However, the basal bacterial scavenging systems are just barely adequate to protect E. coli from endogenous O2- and H2O2 and any circumstances which elevate the rates at which these oxidants enter or are formed inside the cell, overwhelm the defense mechanisms (Imlay 2013). These studies indicate that Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  133  H2O2 contributes in KC-L bactericidal activity which is consistent with the greater susceptibility of kat mutants to this treatment.       In E. coli, addition of exogenous H2O2 or agents producing superoxide (O2-) leads to DNA damage and inhibition of enzymes activities by mismetallation (Imlay 2003; Valco et al. 2005). H2O2 is formed through chemical processes when reduced metal ions and sulfur species are present in oxygenated aqueous environments (Imlay 2008). As H2O2 (unlike O2-) is an uncharged small molecule, it passes membranes at a moderate efficiency comparable to water; thus, intracellular H2O2 stress and oxidative stress are likely to arise in bacteria in H2O2-containing environments, (Imlay 2008, 2013). In fact, mineral surfaces and dissolved metal ions can generate H2O2 through Fenton chemistry (Schoonen et al. 2006; Imlay et al. 1988). Recent studies have revealed the production of H2O2 in some antibacterial clay minerals such as Oregon blue clay (Morrison et al. 2016) and a clay from the Columbian Amazon (Londono et al. 2017).       The studies presented here demonstrated that E. coli mutants deficient in the production of ROS-scavenging enzymes, especially katG and katE, mutants exhibited higher susceptibility to KC-L. E. coli cells are equipped with two catalases (HPI catalase encoded by katG and HPII encoded by katE) that break down H2O2 into H2O and O2, while alkylhydroperoxide reductase (Ahp; encoded by ahpC and ahpF) is reported to provide further defense by reducing various organic hydroperoxides. Two superoxide dismutases (SODs), Mn-containing SOD and Fe-containing SOD, encoded by sodA and sodB respectively, dismutate O2- to H2O2 (Farr 1991).       Moreover, the present study indicates that the E. coli strains deficient in recombination or excision repair strategies such as recA and polA1 (deficient in DNA polymerase 1 (PolI) exhibited higher susceptibility to KC-L. One of the most consequential impacts of oxidative stress in bacteria is mutagenesis, while neither H2O2 nor O2− can directly damage DNA (Imlay 2013). H2O2 is proposed to react with the intracellular pool of unincorporated Fe2+, some of which is associated with DNA, and produce OH• which eventually giving a rise to a wide variety of lesions and ultimate genotoxicity, as       Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  134  E. coli strains that lack recombination (rec) genes are hypersensitive to exogenous H2O2 (Imlay 2008, 2013). Moreover, Solanky and Haydel (2012) studies using Comet assay suggested that DNA damage contributes in the antibacterial activity of CB aqueous leachate. It has been also reported that Al can induce DNA damage and also prevent the repair of DNA lesions in eukaryotic cells (Lankoff et al. 2006).      The results of GFP library provided a real-time gene expression profiling tool for investigating the mechanism of action by offering more insight into transcriptional modulations upon treatment (Onnis-Hayden et al. 2009). KC-L activated DNA repair and SOS regulatory responses induced upon DNA damage in treated cells. The results confirmed the involvement of oxidative stress and SOS responses and more importantly highlighted the complexity of the cellular stress responses such as envelop stress, osmotic shock, acid shock, and biofilm formation induced upon exposure to KC-L. Collectively, this study illustrated the pleiotropic effect of KC-L in simultaneously affecting multiple bacteria targets.           Unique among biological membranes, the OM acts as a permeability barrier due to the presence of LPS and its highly asymmetrical bilayer structure (Helander and Mattila-Sandholm 2000; Ruiz et al. 2006). OM can selectively exclude hydrophobic or relatively large-scaffold antimicrobial agents, detergents, dyes and confers intrinsic resistance in Gram-negative bacteria (Nikaido and Vaara 1985; Denyer and Maillard 2002; Delcour 2009). Importantly, the integrity of the OM can be disturbed by certain substances (collectively called permeabilizers) which weaken the stabilizing interactions among OM structural components (Vaara 1992). Moreover, polycationic antibiotics such as polymyxin B and aminoglycosides interact with the LPS of the OM to enhance its permeability (Rahaman et al. 1998). It has been shown that bacteria can take up large-scaffold antibiotics efficiently, when their OM is perturbed (Muheim et al. 2017).       This study revealed that KC-L acts as an OM permeabilizer agent. Further investigations using single metal ion solutions mimicking the concentration of Al and Fe found in KC-L, showed the permeabilizing impact of Al on OM was comparable to that of the metal ion mixture (MIM). As previously demonstrated, treatment with Al salts induced bacterial membrane damage (Yaganza et al. 2004). Al Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  135  exerts its toxicity through known mechanisms including increasing permeability by binding to the cell wall, replacing divalent metal complexes in membrane or bacterial cells, particularly Mg and Ca, triggering osmoregulative disorders or phosphate deprivation, promoting lipid peroxidation, and binding to DNA (Yaganza et al. 2004; Exley 2004, Oteiza and Verstraeten 2006). The results provided by this study indicate that Al in KC-L can target the bacterial OM and increase its permeability. This in turn can compromise the metal homeostasis in treated bacteria via uncontrolled efflux of metal ions, present in KC-L, at toxic levels, while the presence of metal ions in combination can magnify the exhibited toxicity due to their synergistic effect (Lemire et al. 2013; Gill et al. 2015).      TEM studies provided insight on ultrastructural changes, membrane anomalies, membrane impairment, and leakage of intracellular materials induced upon KC treatment of E. coli and S. aureus as previously used for investigating the mode of action of heavy metal ions and novel antimicrobial agents  (Jung et al. 2008; Lv et al. 2014; Hartmann et al. 2010). However, in morphological studies it is difficult to distinguish whether membrane disruption or cell wall detachment is a cause or a consequence of bacterial death (Lemire et al. 2013). Thus, we further studied CM integrity as it can be severely compromised upon exposure to toxic levels of metals such as Al and Ag (Yaganza et al. 2004; Jung et al. 2008; Feng et al. 2000) as well as oxidative-stress damage to membrane which may arise via either lipid peroxidation or deleterious damage to membrane proteins (Farr and Kogoma 1991; Ezraty et al. 2017). Several membrane-damaging agents may interact with multiple targets via disruption of membrane structural and functional integrity, through steric inhibition of membrane-embedded proteins and/or by alteration of the proton motive force, which eventually may lead to leakage of intracellular content and bacterial death (Hurdle et al. 2011; Straus and Hancock 2006). The ability of bacteria to acquire resistance to such membrane-active agents is restricted. Furthermore, enzyme mismetallation has been identified as a critical consequence of both metal intoxication and peroxide stress (Imlay 2014).       These data together indicate that the bactericidal activities associated with KC-L can be, at least in part, due to ROS-mediated bacterial damage in which metal-induced oxidative reactions could underlie Chapter 5: Elucidating the Mode(s) of Action of KC Leachates  136  membrane or DNA damage. In addition, the synergistic effect of metal ions together with the membrane impairment caused by metals such as Al can exacerbate generation of ROS. Collectively, these results suggest a complex multi-target mode of action for KC-L that leads to lethal pleiotropic effects in treated bacteria. Better understanding the KC’s mode of action and bacterial survival strategies opens up new avenues for devising clay mineral-based antibacterial strategies. 137  Conclusions and Future Directions The pressing need for novel antimicrobial agents and innovative strategies      Given the importance of natural and synthetic antimicrobial agents in modern medicine and the declining arsenal of antimicrobials to combat MDR bacteria, there is an increasing apprehension over the threat of antibiotic resistance and a pressing need for new antimicrobial agents. No novel broad-spectrum antibiotic has been developed for several decades and despite intensive searches for new antimicrobials, there are few active candidates in the pipeline. For decades, considerable attention has been focused on natural products as an invaluable source of novel therapeutic agents and bioactive compounds. However, they have mostly been overlooked due to a lack of understanding of their complex nature and mode of action. In addition, they primarily required rigorous scientific analysis to identify their active principal components in an effort to define their controlled efficacy. This study provides the first comprehensive analysis of Kisameet clay (KC), a naturally-occurring clay mineral from British Columbia, Canada, and describes its in vitro broad-spectrum antimicrobial properties against a variety of MDR and XDR clinical isolates.  The complex nature of KC      The natural heterogeneity found in the KC deposit emphasizes the vital role of physico-chemical and biological analysis of harvested clay mineral samples for further therapeutic applications. It appears that, among antibacterial KC samples, exchange of metal ions, pH, buffering capacity, and redox potential are the critical features of the antibacterial activity compared to the mineralogical composition. In addition, most of the antibacterial KC samples were harvested from the surface (depth 0-4 ft) of the deposit. Therefore, further analysis of the surface water in parallel to the clay samples is vital to the identification of the environmental factors that affect KC’s characteristics. Moreover, preparation of meta datasets based on all the characterized properties in this study might facilitate further searches for antibacterial clay samples from the deposit. Chapter 6: Conclusions and Future Directions 138       The KC deposit, as a kind of extreme environment, harbors an unexpectedly rich community of bacteria; metagenomic studies (Svensson et al. 2017) along with culture-based isolation of resident bacteria presented in this study, have provided a valuable insight into the KC resident bacteria and the potential for further investigation of their economical or environmental importance. Notably, 25% of KC resident bacteria isolated in this study belong to the class of Actinobacteria, which includes Streptomyces and Arthrobacter species as well-known producers of antibiotics and secondary metabolites. Natural products have played vital roles in antibiotic discovery and development; specifically, large number of soil Actinobacteria account for 70-80% of commercially available secondary metabolites as well as 50% of known antibiotics. Thus, further investigation of the KC isolates in terms of their potential as producers of novel bioactive compounds may uncover valuable compounds as KC and its composition is distinct from soil. Moreover, it remains to be investigated whether the KC resident bacteria (especially isolates belonging to Pseudomonas species) can tolerate the antibacterial KC samples under the in vitro conditions implemented in this study, which may provide knowledge about potential resistance mechanisms against KC and/or antibacterial clay minerals. Future studies integrating metagenomic approaches together with proteomic technology will be vital to explore the resistome of this deposit as an ancient metal-rich clay deposit as well as the discovery of novel small molecules.       Broad-spectrum antimicrobial properties of KC      The results provided in this study revealed that KC exhibits an in vitro broad-spectrum antibacterial activity against bacterial laboratory strains and pathogenic isolates responsible for acute or chronic infections, under the conditions described. KC showed potent bactericidal effect against a panel of MDR ESKAPE pathogens, which are responsible for the majority of recalcitrant bacterial outbreaks in nosocomial settings, while the therapeutic choices are extremely limited. Moreover, KC exhibited potent antibacterial activity against a collection of MDR and XDR clinical isolates of BCC, P. aeruginosa, and S. maltophilia isolated from CF patients as some of the most challenging bacterial pathogens.Chapter 6: Conclusions and Future Directions  139       Members of the BCC are responsible for the most challenging of all persistent pulmonary infections in CF patients due to their remarkable resistance to the available therapeutic agents. As current antimicrobial options for BCC are limited and antimicrobial resistance evolves rapidly, development of novel therapeutic strategies aimed to control BCC bacteria and other MDR infections in CF patients are required desperately. This study demonstrates the antibacterial effect of KC against all CF clinical isolates tested including sequential isolates from chronic infections of B. cenocepacia and B. multivorans, the two most common BCC species accounting for around 85% of all BCC infections, and P. aeruginosa isolates as the most common pathogen in CF patients. These data extend our previous findings on the potent bactericidal effect of KC against MDR clinical isolates of P. aeruginosa among ESKAPE pathogens. My studies have indicated the significant susceptibility of all P. aeruginosa strains tested, laboratory strain, clinical isolates, and epidemic pathogens to both KC and KC-L. The results presented here suggest that further investigation of other globally important Burkholderia pathogens such as B. pseudomallei would be valuable. An outstanding question that remains to be answered is whether KC may be a potential therapeutic option, as a complementary or a suppressive antimicrobial treatment, for pathogenic colonization and chronic pulmonary infections in CF patients and also in cases of CGD. As inhalation antibiotic therapy is now commonly used to deliver high concentrations of medicine to the lung, KC-L or its derivatives might have the potential of being developed and employed as an aerosolized drug administration.       KC is also active against C. albicans and C. neoformans as two major human fungal pathogens. Modern medicine has paradoxically increased the number of immunocompromised patients who are more vulnerable to fungal infections named as “hidden killers”. The effectiveness of KC-L on these fungal pathogens still awaits evaluation. Moreover, recent studies indicate that resistance is more a property of microbial communities than that of an individual pathogen, which in turn highlights the crucial role of multi-microbial context during infections. In addition, the remarkable resistance of biofilms (multicellular Chapter 6: Conclusions and Future Directions  140  communities of microorganisms) to antimicrobial agents has been well documented. Thus, whether KC or KC-L can inhibit biofilms composed of a mixture of microbial communities remains to be investigated.       The Heiltsuk First Nation has employed KC in geophagia for a variety of internal ailments, suggesting that this natural mineral might be an option for treatment of intractable infections such as Clostridium difficile. It remains to be seen how KC or its derivatives may affect the intestinal microbiome upon treatment. Moreover, whether KC acts synergistically with common antimicrobials to potentiate or enhance their activity against resistant pathogens could be another interesting research topic. Despite a long history of KC medicinal applications, to date, no toxic side effects have been reported in human use of KC. However, scientific investigation of the health influences due to prolonged applications at various concentration levels, detailed in vivo studies in animal models, as well as cytotoxicity studies remain to be carried out.   Metal ion toxicities in a pH-dependent manner      The results obtained in this study support the initial hypothesis about the critical role of exchangeable transition metal ions in a pH-dependent manner in the antibacterial activity of KC. Nonetheless, further investigations have provided evidence about the important role of Al content. Many of the transition metal ions are essential metals with structural and functional roles in microorganisms, while their fundamental roles are likely to be underestimated as not all have been fully characterized. For decades, considerable numbers of investigations have focused on metal resistance; while there have been limited attention on the biochemical or biophysical modes of action by which specific metals exert toxicity toward microorganisms. Nevertheless, a better understanding of metal homeostasis along with microbial metabolic diversities, are inevitably imperative to improve the design of effective metal-based antimicrobial therapies to combat recalcitrant pathogens.       Treating bacteria with KC-L, rich in metal ions such as Al, Fe, Co, Cu, Ni, and Zn at µM to mM concentrations causes a sudden challenge in maintaining their metal homeostasis. Mismetallation of Chapter 6: Conclusions and Future Directions  141  metalloenzymes is an inevitable aspect of such a condition in a hyper-metallic aqueous environment. Although bacteria have evolved mechanisms to protect their cellular functions from metal intoxication, the function of high-affinity uptake systems rapidly cause metal intoxication which is exacerbated by the absence of allosteric feedback inhibition for most metal uptake systems. It seems that bacterial evolution in environments with limited soluble/available essential metals has focused on metal acquisition more efficiently rather than quick metal intoxication. This point can be an Achilles’ Hill for treatment of pathogenic bacteria with metal-restricted habitats.        Metal ions can discriminate between prokaryotic and eukaryotic cells, as they handle the uptake of metals with divergent metal transport systems and metalloproteins; however, non-essential metals can enter cells non-specifically or via misuse of essential-metal transporters (i.e. Al3+ using the Fe3+ uptake system in bacteria). In addition, there is a gap in current knowledge concerning the uptake of some non-essential metals, which warrants attention. Cell culture studies need to be carried out to detect any associated damage upon KC-L or its derivative treatments in order to apply such antimicrobial agents in medical applications.          The Irving-Williams series of ligand affinity, demonstrating the affinity of essential divalent metal ions toward biomolecules, indicates how high concentrations of essential metal ions such as Cu2+, Zn2+, Ni2+, Co2+, and Fe2+ can displace correct metals during the compromised metal homeostasis. Waldron and Robinson (2009) have described this concept that the Irving-Williams series may provide more insight into bacterial vulnerability toward metals guiding the preparation of more effective MBA. Our results together with this perspective and a proactive approach toward metal resistance can guide development of improved formulations of artificial KC-L as an effective formulation. For instance, the high sensitivity of P. aeruginosa (PAO1, MDR isolates, and XDR epidemic strains) toward KC leachate and metal ions might be a guide to use alternative metal-based antimicrobial compounds in the battle against this major recalcitrant pathogen. While the potent inhibitory action of KC against a broad-spectrum of bacterial pathogens represents a promising alternative area of research, for the design of novel antimicrobial agents Chapter 6: Conclusions and Future Directions 142 from natural resources, the further investigation of metal ion contents of KC-L, as presented in this study, provides a specific direction to assess the therapeutic potential of defined artificial preparations for the inhibition of microbial growth.       Contrary to most of antibiotics with known target, metals prompt bactericidal efficacy through multiple modes; thus, theoretically simultaneous multiple mutations within a bacteria are required to elicit any resistance. Although the potent and multi-targeted modes of action of KC-L restrict the likelihood of resistance development, and to date no resistance to KC has been observed, further more specific studies to elucidate how bacteria may develop resistance to KC and what mechanisms are involved remain to be carried out. On the other hand, the effectiveness of KC-L against bacterial strains with known resistance to metals still awaits evaluation.   OM permeabilization and CM impairment upon KC leachate treatment      The results presented in this study reveal that KC-L disrupts the integrity of the OM and permeabilizes it in E. coli. Undoubtedly, many of the recalcitrant superbugs are Gram-negative species (i.e. four members of the ESKAPE pathogens), against which development of efficacious novel therapies is challenging, partly due to their OM intrinsic permeability barrier functions to exclude a variety of antimicrobial agents. Certain substances that weaken the stabilizing interactions between OM constituents (i.e. cation binding sites of LPS), either by releasing OM components or by disorganizing the whole OM (i.e. polycationic antimicrobials complex avidly with LPS in the OM) are collectively called permeabilizers.           Efficient permeabilizers of the OM can improve the arsenal of antibiotics’ effectiveness due to their ability to sensitize Gram-negative bacteria to ineffective hydrophobic or large-scaffold existing antibiotics via enhancing their ability to access their bacterial targets. It warrants attention and future investigation to see if KC-L sensitizes Gram-negative bacterial pathogens to antimicrobial agents whose impermeability through OM may restrict their efficacy or the spectrum of clinical applications. For any combination, Chapter 6: Conclusions and Future Directions  143  however, the impact of KC-L chemical components on the bioavailability and/or efficacy of therapeutic compounds should be considered in order to maximize the collateral efficacy. An outstanding question is how KC-L affects the OM, whether through displacement or extraction of divalent cations from this membrane and eventual destabilization and permeabilization of it to KC-L components in a process termed “self-promoted uptake”, as described previously for polycationic antimicrobials such as polymyxins and cationic peptides (Hancock and Chapple 1999; Zhang et al. 2000).      While TEM studies provided more insight into the ultrastructural changes upon KC-L treatment, further TEM studies of thin sections of resin-embedded treated bacteria may detect more specific ultrastructural modifications such as vacuole formation, cytoplasm condensation, abnormalities and disorganization in the cell wall including thinning, budding, or thickening, separation of the CM from the OM, disorganization of CM, disintegration of the cell wall, crack, rupture, electron dense particles or precipitates in treated cells with different preparations of KC-L compared with that of metal ion solutions.    Multi-target mechanism of action       The information obtained in this study suggests that the bactericidal activity of KC-L is due to multiple modes of action. The low-pH buffered environment rich in a combination of released metal ions can synergistically challenge treated bacteria to control efflux of metal ions and maintain their metal homeostasis, while Al3+ -related impairment of OM integrity may exacerbate this situation. Through the complex metal mixture at low-pH and high concentration, KC-L can concurrently stress multiple bacterial components, cause metal intoxication and consequential cellular damage, impair and destabilize OM and CM, cause oxidative stress, and produce H2O2, which collectively lead to lethal pleiotropic effects in treated bacteria.       To assess the role of oxidative stress, I did not use dye-based ROS detection methods such as application of reduced fluorescein and rhodamine dyes as ROS probes, as it has been reported that the disruption of membrane can impact the amount of dye penetrating into bacterial cells and result in Chapter 6: Conclusions and Future Directions 144 misinterpretation (Imlay 2015). For more in depth understanding, a specific study of OxyR, as the natural sensing mechanism of cells by which the threatening levels of H2O2 can be detected, remains to be carried out. Further work also could explore in more detail the production of H2O2 and its concentration and to see whether its generation together with low pH in the metal-rich environment of KC-L contribute in the cidal activity.       This study demonstrated bacterial DNA damage upon KC-L treatment under the in vitro condition described. However, the precise mechanism(s) of DNA damage is not clear and whether it is linked to Fe2+-mediated Fenton chemistry, the metal toxicity related to Co2+ or Mn2+, to oxidative stress, or combined effects remains to be elucidated. Further comparative investigations with metal ion solutions, as described in this study, could explore it in more details. The realization that KC-L simultaneously interacts with multiple bacterial targets may profoundly modify and guide drug design and medicinal application. Furthermore, defining the mode(s) and molecular machinery involved in the inhibitory action of KC toward pathogenic MDR bacteria has the potential to uncover novel therapeutic targets for the treatment of infections.      While the results presented here indicate that the antibacterial activity of KC-L occurs through the soluble metal fraction using E. coli as a model organism, it is worth pointing out that these findings do not exclude the potential role of KC mineral particles to exert inhibitory or combinatory action to that of the soluble fraction of KC suspensions. Further studies of mineral particles, more specifically the size-based fraction of sequentially washed clay particles to remove the soluble or exchangeable ions may provide more insight into the antibacterial properties of nanoparticles inherent in KC about which much less is known. Naturally-occurring clay minerals have been considered eco-friendly products for centuries; however, with respect to their metal-rich nature, perhaps sustainability practices of their restricted, indiscriminate applications and their wastes should be considered.  Chapter 6: Conclusions and Future Directions 145 Significance of this study      As rapid bacterial evolution toward resistance has overwhelmed traditional antibiotic defenses, natural alternatives such as clay minerals are receiving attention for their potential antimicrobial efficacy and applications. Whilst natural clay minerals are heterogeneous mixtures, defining the specific characteristics required for antimicrobial activity is essential in order to improve their chemical consistency and standardize their medicinal applications. The results from this study represent the first comprehensive attempt to elucidate the microbicidal properties of KC, demonstrating the broad-spectrum antimicrobial activities of KC and its derivatives in vitro, and its chemical and biological properties involved in this action. In addition, by integrating physico-chemical characterizations together with microbiological studies, herein, the complex antibacterial activities of KC have been dissected from multiple aspects. The data provided by this study could be a potential source for defining and controlling the antibacterial activity of this natural clay as well as its derivatives for medicinal application. 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Ann Am Thorac Soc 12:70-78.  http://doi.org/10.1513/AnnalsATS.201408-395OC.173 Appendices Appendix A  A.1 Culture Media Composition  To isolate KC resident bacteria, a variety of culture mdedida, as listed in section 2.2.7 were used. Here, their components (g L-1) are as follows:  Hickey-Tresner Agar (HTA)Dextrin, 10.0; pancreatic digest of casein, 2.0; meat extract, 1.0; yeast extract, 1.0; CaCl2, 0.002; agar, 15.0; (pH: 7.2 +/- 0.2)  Actinomycete Isolation Agar (AIA)Sodium propionate, 4.0; sodium caseinate, 2.0; K2HPO4, 0.5; asparagine, 0.1; MgSO4.7H2O, 0.1; FeSO4.7H2O, 1.0; glycerol, 5.0 mL; agar, 15.0; (pH: 8.2 +/- 0.2)  Inorganic Salt-Starch (ISP-4) mediumSoluble starch, 10.0; K2PO4, 1.0; MgSO4.7H2O, 1.0; NaCl, 1.0; (NH4)2SO4, 2.0; CaCO3, 2.0; trace salts solution, 1 mL; agar, 20.0; (pH: 7.0-7.4). Trace salts solution includes FeSO4.7H2O, 1.0; MnCl2.4H2O, 1.0; ZnSO4.7H2O, 1.0.  M1 mediumSoluble starch, 10.0; yeast extract, 4.0; peptone, 2.0; agar, 20.0; in natural seawater (Mincer et al. 2005).  OA medium (phosphate-buffered, 0.01M)Fe(III)-EDTA, 10 µM; DL-malate, 2.0; Na-acetate, 3.0;  NH4Cl, 10.0; Macro salts [MgSO4.H2O, 2.0; CaCl2, 0.1], trace elements [borate; molybdate; Zn; Mn; Cu; Co], and vitamin supplements [thiamine, Appendices 174 10.0; biotin, 15.0; niacin, 100.0; vitamin B12, 200.0; p-aminobenzoic acid, 50.0 (mg/100 mL of dH2O)], 1 mL L-1; (pH 6.8) A.2 Bacterial Strains Isolated From KC Samples Table A.1 KC isolated identified by 16SrRNA sequencing No. Name KC sample Media KC islates based on 16S rRNA sequences 1 SB1 KC35 HTA  Arthrobacter spp. 2 SB2 KC35 HTA Arthrobacter spp. 3 SB3 KC35 HTA or AIA Arthrobacter spp. 4 SB6 KC35 HTA or AIA Arthrobacter spp. 5 SB8 KC35 HTA  Cellulomonas spp.  6 SB9 KC35 HTA Arthrobacter spp.  7 SB11 KC14 Pea peptone Bacillus spp. 8 SB12 KC14 Soy  peptone Sporosarcina spp. 9 SB13 KC14 TSB Paenibacillus spp. 10 SB14 KC14 Lupin peptone Arthrobacter spp.  11 SB15 KC14 Potato peptone Psychrobacillus spp. 12 SB16 KC14 Lupin peptone Bacillus spp.  13 SB17 KC14 Pea peptone  Psychrobacillus spp. 14 SB19 KC14 Lupin peptone Bacillus spp.  15 SB20 KC14 Potato peptone Bacillus spp.  16 SB21 KC14 Soy peptone  Sporosarcina soli 17 SB22 KC14 Soy peptone  Sporosarcina spp. 18 SB23 KC14 Soy peptone Sporosarcina spp. 19 SB24 KC14 N-Z amine Paenibacillus spp. 20 SB25 KC35 N-Z amine Rhodanobacter spp. 21 SB26 KC14 Lupin peptone Arthrobacter spp.  22 SB27 KC35 N-Z amine Rhodanobacter spp. 23 SB28 KC14 Lupin peptone Bacillus spp. 24 SB29 KC14 TSB Sporosarcina spp. 25 SB30 Kis5-12 TSA Paenibacillus spp. 26 SB31 Kis5-12 HTA Pseudomonas spp. 27 SB32 Kis5-12 TSA Pseudomonas spp. 28 SB33 Kis5-12 HTA Pseudomonas stutzeri 29 SB34 Kis5-12 HTA Chryseobacterium spp. 30 SB35 Kis5-12 TSA Pseudomonas stutzeri 31 SB36 Kis5-12 TSA Pseudomonas stutzeri 32 SB37 Kis5-12 TSA Dietzia spp. Appendices 175 No. Name KC sample Media KC islates based on 16S rRNA sequences 33 SB38 Kis5-12 TSA Achromobacter pestifer 34 SB39 Kis5-12 HTA Bacillus spp. 35 SB40 Kis5-12 HTA Pseudomonas stutzeri 36 SB41 Kis5-12 TSA Pseudomonas stutzeri 37 SB42 Kis5-12 TSA Pseudomonas stutzeri 38 SB43 KC14 Potato peptone Bacillus spp. 39 SB44 KC14 N-Z amine Bacillus spp. 40 SB45 KC14 Pea peptone Sporosarcina spp. 41 SB46 KC14 Pea peptone Sporosarcina spp. 42 SB47 KC14 Potato peptone Bacillus spp. 43 SB48 KC14 Soy peptone Sporosarcina spp. 44 SB49 Kis5 ISP4 Exiguobacterium spp. 45 SB50 KC35 Soy peptone Arthrobacter spp.  46 SB51 KC35 Pea peptone Rhodanobacter spp. 47 SB52 KC14 Potato peptone Psychrobacillus spp. 48 SB53 KC35 Pea peptone Rhodanobacter spp. 49 SB54 KC14 Wheat peptone Rhodanobacter spp. 50 SB55 KC14 Potato peptone Psychrobacillus spp. 51 SB56 KC14 Potato peptone Bacillus spp. 52 SB57 KC14 Potato peptone Bacillus spp. 53 SB58 KC14 Potato peptone Bacillus spp. 54 SB59 KC14 Potato peptone Bacillus spp. 55 AM1 Kis5-28 ISP4 Stenotrophomonas rhizophilia 56 AM3 Kis5-28 ISP4 Lysinibacillus spp. 57 AM5 Kis5-28 ISP4 Arthrobacter spp. 58 AM6 Kis5-28 ISP4 Oerskovia spp. 59 AM7 Kis5-24 ISP4 Sphingobacterium spp. 60 AM8 Kis5-24 ISP4 Brevundimonas spp. 61 AM9 Kis5-24 ISP4 Sphingobacterium spp. 62 AM10 Kis5-4 ISP4 Lysinibacillus spp. 63 AM11 Kis5-20 ISP4 Sphingobacterium spp. 64 AM12 Kis5-20 ISP4 Pseudomonas stutzeri  65 AM14 Kis5-16 ISP4 Stenotrophomonas rhizophila 66 AM17 Kis5-12 ISP4 Collimonas spp. 67 AM18 Kis5-8 ISP4 Achromobacter pestifer 68 AM21 Kis5 ISP4 Lysinibacillus spp. 69 AM22 Kis5 ISP4 Lysinibacillus spp. 70 AM23 Kis5 ISP4 Lysinibacillus spp. 71 AM31 Kis5-8 ISP4 Janthinobacterium spp.  72 AM32 Kis5-4 ISP4 Enhydrobacter spp. Appendices 176  No.  Name KC sample Media KC islates based on 16S rRNA sequences 73 AM33 Kis5-12 ISP4 Pseudomonas spp. 74 AM35 Kis5-28 ISP4 Arthrobacter spp. 75 AM36 Kis5-28 ISP4 Oerskovia spp. 76 AM37 Kis5-28 ISP4 Arthrobacter spp. 77 AM38 Kis5-28 ISP4 Arthrobacter spp. 78 AM40 Kis5-28 ISP4 Arthrobacter spp. 79 AM42 Kis5-28 ISP4 Stenotrophomonas rhizophila 80 AM43 Kis5-28 ISP4 Stenotrophomonas rhizophila 81 AM44 Kis5-28 ISP4 Arthrobacter spp. 82 AM45 Kis5-28 ISP4 Oerskovia spp. 83 AM46 Kis5-28 ISP4 Stenotrophomonas rhizophila 84 AM47 Kis5-28 ISP4 Oerskovia spp. 85 AM48 Kis5-28 ISP4 Oeroskovia spp. 86 AM49 Kis5-28 ISP4 Oerskovia spp. 87 AM50 Kis5-24 ISP4 Pseudomonas spp. 88 AM51 Kis5-16 ISP4 Pseudomonas spp. 89 AM52 Kis5-12 ISP4 Pseudomonas spp. 90 AM56 Kis5-8 ISP4 Streptomyces spp. 91 AM57 Kis5-24 ISP4 Rhodococcus erythropolis 92 AM58 Kis5-24 ISP4 Oerskovia spp. 93 AM59 Kis5-24 ISP4 Microbacterium spp. 94 AM60 Kis5-24 ISP4 Brevundimonas spp. 95 AM61 Kis5-24 ISP4 Exiguobacterium spp. 96 AM66 Kis5-8 M1 Pseudomonas putida 97 AM70 Kis5-16 M1 Pseudomonas spp. 98 AM71 Kis5-16 M1 Pseudomonas spp. 99 AM73 Kis5-20 M1 Pseudomonas spp. 100 AM80 Kis2-32 ISP4 Pseudomonas spp. 101 AM82 Kis2-32 ISP4 Pseudomonas spp. 102 AM84 Kis5-16 M1 Microbacterium spp. 103 CCH1 Kis5-12 HTA Rhodococcus erythropolis 104 CCH2B Kis5-12 HTA Rhodococcus erythropolis 105 CCH13 Kis5-12 HTA Stenotrophomonas rhisophila 106 CCH14 Kis5-12 HTA Stenotrophomonas rhisophila 107 CCH17 Kis5-12 HTA Herminiimonas spp. 108 CCT1 Kis5-12 TSA Pseudomonas spp. 109 CCT17 Kis5-12 TSA Microbacterium spp. 110 CCT18 Kis5-12 TSA Pseudomonas spp. 111 SS1 Kis3-0 OA Pseudomonas spp. 112 SS2 Kis3-0 OA Pseudomonas putida Appendices 177 No. Name KC sample Media KC islates based on 16S rRNA sequences 113 SS4 Kis3-0 OA Pseudomonas spp. 114 SS5 Kis3-0 OA Bacillus spp. 115 SS6 Kis3-0 OA Bacillus spp. 116 SS7 Kis3-0 OA Bacillus spp. 117 SS8 Kis3-0 OA Bacillus spp. HTA: Hickey-Tresner agar; AIA; Actinomycete isolation agar; TSB and TSA: Tryptic soy broth or agar, respectively Appendices 178 Appendix B  B.1 Metal Stability Constants of Cation Chelators Table B.1 Metal stability constant (pKs) of cation chelators (The information adapted from Makrlik and Vanura 1992; Day and Ackrill 1993; Kontoghiorghes 1995; Keberle 1964; and Flora and Pachauri 2010). Metal ion EDTA DFO BPY Al3+16.1 22.0 - Ca2+10.6-11.0 2.0 - Cd2+16.4 - - Co2+ 16.1 11.0 16.1 Cr3+ - 21.0 - Cu2+18.4 14.0 17.5 Fe2+14.4 10.0 17.5 Fe3+25.1 30.6-31 - Mg2+8.7-9.0 4.0 - Mn2+13.4 - - Ni2+18.4 10.0 20.1 Pb2+18.3 - - Zn2+16.1-16.6 11.0-11.1 13.7 Appendices 179 Appendix C  C.1 Sensitivity of E. coli Mutants with Defect in Iron-Regulation or -Storage Functions to KC Leachate Table C.1 E. coli bacterial strains with mutations in iron-regulation or iron-storage genes No. Name Mutant Genotype/characteristics Reference1 JW0669-2 fur As BW25113 plus ΔlacZ4787(::rrnB-3), Δfur-731::kan Baba et al. 2006 2 JW3298-1 bfr As BW25113 plus Δbfr-746::kan Baba et al. 2006 3 JW0797-1 dps As BW25113 plus Δdps-784::kan Baba et al. 2016 4 JW1893-1 ftnA As BW25113 plus Δftn-755::kan  Baba et al. 2006 5 JW1890-1 ftnB As BW25113 plus ΔftnB752::kan Baba et al. 2006 All the strains are congenic derivatives of E. coli BW25113 (WT) and obtained from Yale University, KEIO collection (Baba et al. 2016). E. coli contains at least four genes that may play roles in iron storage: bfd, encoding the [2Fe-2S]-containing bacterioferritin-associated ferredoxin (Bfd), and bfr, encoding bacterioferritin (Bfr), and ftnA encoding a ferritin (FtnA), and ftnB, encoding a ferritinlike protein (FtnB). Thus, the homeostasis of iron ions is tightly regulated so that their intracellular concentrations do not reach toxic levels (Andrews et al. 2003; Abdul-Tehrani et al. 1999). It has been reported that intracellular Fe can be stored in addition to iron detoxification proteins (Dps) which are employed to protect the bacterial DNA from iron-induced free radical damage during oxidative stress (Imlay 2015). Dps is a member of H2O2-stress regulons while Fur is an iron-import control (Andrews et al. 2003; Imlay 2015). As our studies previously revealed the iron-rich nature of KC-L, the effect of mutation in iron regulation and storage genes was studied using E. coli mutants (Table C.1). Fig. C.1 summarized the results. Appendices 180 0 4 8 1 2 1 6 2 0 2 40123456789fu rT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100furfur+ dH2Ofur+ L1000 4 8 1 2 1 6 2 0 2 40123456789b frT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100bfrbfr+ dH2Obfr+ L1000 4 8 1 2 1 6 2 0 2 40123456789d p sT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100dpsdps+ dH2Odps+ L1000 4 8 1 2 1 6 2 0 2 40123456789f tn AT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100ftnAftnA+ dH2OftnA+ L100A B C D Appendices 181 0 4 8 1 2 1 6 2 0 2 40123456789ftnBT im e  (h )Log10 CFU/mLWTWT+ dH2OWT+ L100ftnBftnB+ dH2OftnB+ L100  Figure C.1 Sensitivity of mutants with defects in iron regulation and storage related functions to KC-L (L100). The dotted line at log10=1 of the Y axis represents the limit of detection for CFU. Error bars represent the standard error (SE) of the mean of at least three independent replicates. Antibacterial activity of L100 against mutant strains was compared to that of parental strain, E. coli BW25113, in MBMM defined medium E Appendices 182 Appendix D  D.1 Description of GFP Clones of E. coli MG1655 Summarized in Fig. 5.11. Table D.1 Function of GFP clones summarized in Fig. 5.11. No. Promoter Description 1 ais protein induced by aluminum, phosphoglycerate mutase-like domain 2 sodB superoxide dismutase, iron 3 recN protein used in recombination and DNA repair (2nd module) 4 ybfE LexA regulated, possible SOS response (upregulated by exposure to DNA-damaging agents)  5 yrbA putative transcriptional regulator (BolA family) ( responsive to osmotic stress)  6 yjiW LexA regulated, possible SOS response 7 dinB DNA polymerase IV, devoid of proofreading, damage-inducible protein P (1st module) 8 napF Fe-S ferredoxin-type protein: electron transfer 9 ompN outer membrane protein N, non-specific porin (1st module) 10 upgB ABC superfamily (peri_bind)  sn-glycerol 3-phosphate transport protein (2nd module) 11 yiiP putative CDF family transport protein 12 ynaI putative transmembrane protein 13 dctA DAACS family,  C4-dicarboxylic acids transport protein 14 betT BCCT family, high-affinity choline transporter (2nd module) 15 ycaD putative MFS family transport protein (1st module) (transporter protein) 16 dppA ABC superfamily (peri_bind)  dipeptide transport protein (1st module) (transporter protein) 17 ycaK putative electron transfer flavoprotein-NAD/FAD/quinone oxidoreductase 18 yicE putative CPA1 family, sodium:hydrogen transport protein (1st module) 19 ftsQ essential cell division protein FtsQ 20 potF ABC superfamily (peri_bind)  putrescine transporter (1st module) 21 ydbD putative oxidoreductase, aldo/keto reductase family, NAD(P)-linked (2nd module) 22 fepB ferric enterobactin ABC transporter 23 b1240 unknown CDS 24 ycjG putative chloromuconate cycloisomerase (muconate cycloisomerase) (2nd module) 25 soxR soxR transcriptional dual regulator 26 fur transcriptional  repressor of iron transport  (Fur family) 27 yahD conserved hypothetical protein 28 marR transcriptional repressor for antibiotic resistance and oxidative stress (stress response) 29 bacA bacitracin resistance; possibly phosphorylates undecaprenol 30 dinJ damage-inducible protein J 31 yebG DNA damage-inducible gene in SOS regulon, dependent on cyclic AMP and H-NS Appendices 183 No. Promoter Description 32 bo374 flagellar protein; similar to 3rd module of ATP-binding components of transporters 33 katE catalase; hydroperoxidase HPII(III) , RpoS dependent 34 yfiE putative transcriptional regulator (LysR family) 35 yaiZ unknown CDS 36 allS putative transcriptional regulator LYSR-type 37 ymfE e14 prophage 38 ybaK conserved hypothetical protein 39 emrE DLP12 prophage; MFP family auxillary multidrug transport protein, methylviologen and ethidium resistance 40 trmE GTP-binding protein with a role in modification of tRNA 41 emrA multidrug resistance secretion protein 42 insA-1 IS1 protein InsA 43 ahpF alkyl hydroperoxide reductase subunit, FAD/NAD(P)-binding; detoxification of hydroperoxides (2nd module) 44 dsbG periplasmic disulfide isomerase, thiol-disulphide oxidase (1st module) 45 nhaA NhaA family of transport protein, Na+/H antiporter (1st module) 46 sodC superoxide dismutase precursor (Cu-Zn) 47 cbpA curved DNA-binding protein 48 borD DLP12 prophage; bacteriophage lambda Bor lipoprotein homolog, involved in serum resistance 49 marC inner membrane protein involved in multiple antibiotic resistance 50 pmrD polymyxin resistance protein B 51 lolA periplasmic protein effects translocation of lipoproteins from inner membrane to outer membrane 52 slyA transcriptional activator for hemolysin (MarR family) 53 b0373 putative transposase-related protein 54 hscB Hsc20 co-chaperone that acts with Hsc66 in IscU iron-sulfur cluster assembly 55 sanA vancomycin sensitivity, putative oxidoreductase 56 mdtH putative MFS superfamily transport protein 57 malF ABC superfamily (membrane) maltose transport protein (2nd module) 58 oxyR transcriptional regulator of oxidative stress, regulates intracellular hydrogen peroxide (LysR family) 59 leuA 2-isopropylmalate synthase60 flgM anti-FliA (anti-sigma) factor; also known as RflB protein 61 araE MFS family,  L-arabinose: proton symport  protein (low-affinity transporter) (1st module) 62 rpoH sigma H (sigma 32) factor of RNA polymerase; transcription of heat shock proteins induced by cytoplasmic stress 63 rhaT DMT Superfamily, L-rhamnose:H+ symporter protein (1st module) 64 ligB putative DNA ligase 65 fliA sigma F (sigma 28) factor of RNA polymerase, transcription of late flagellar genes (class 3a and 3b operons) 66 ompR transcriptional regulator in two-component regulatory system with EnvZ, affects OM protein sythesis (OmpR) 67 hlyA hemolysin E 68 rpoE sigma E (sigma 24 ) factor of RNA polymerase, response to periplasmic stress (TetR/ArcR family) 69 ftsZ tubulin-like GTP-binding protein and GTPase, forms circumferential ring in cell division 70 recA DNA strand exchange and recombination protein with proteiase and nuclease activity (1st module) Appendices 184 No. Promoter Description 71 fliF flagellar biosynthesis; basal-body MS(membrane and supramembrane)-ring and collar protein 72 polB DNA polymerase II and and 3' --> 5' exonuclease 73 arcB multimodular ArcB: membrane part of sensory histidine kinase in two-component regulatory system with ArcA 74 galP MFS family, galactose:proton symporter (1st module) 75 uvrD DNA-dependent ATPase I and helicase II (1st module) 76 htpG chaperone Hsp90, heat shock protein C 62.5 77 lon DNA-binding, ATP-dependent protease la; cleaves RcsA and SulA, heat shock k-protein (ATP ase activity)  78 phoB response regulator in two-component regulatory system with  PhoR (or CreC) ,regulates  Pi uptake (OmpR)  79 fliE flagellar biosynthesis; basal-body component 80 gyrB DNA gyrase, subunit B (type II topoisomerase) (1st module) 81 greB transcription elongation factor and transcript cleavage 82 lexA transcriptional repressor for SOS response (signal peptidase of LexA family) 83 katG catalase; hydroperoxidase HPI(I) 84 ssb ssDNA-binding protein controls activity of RecBCD nuclease (stress response) 85 yfcV putative fimbrial-like protein 86 sseB enhances serine sensitivity 87 soxS transcriptional activator of superoxide response regulon (AraC/XylS family) 88 mscS putative membrane protein,  involved in stability of MscS mechanosensitive channel, (1st module) 89 rhlE putative ATP-dependent helicase (2nd module) 90 ybjK putative transcriptional regulator (DeoR family) 91 ydjN putative transport protein (1st module) 92 ybjJ putative transport protein/putative regulator (1st  module) (1st module) 93 ykgJ putative ferredoxin 94 asr acid shock protein 95 insA-7 IS1 protein InsA (stress response)  96 rnk regulator of nucleoside diphosphate kinase 97 recX regulator, (SOS response/DNA repair) 98 citC citrate lyase synthetase (citrate (pro-3S)-lyase ligase) 99 glnK regulatory protein, P-II 2, nitrogen assimilation by glutamine synthetase, regulates GlnL (NRII) and GlnE (ATase) 100 gcl glyoxylate carboligase 101 yfbS putative response regulator (1st module) 102 ygcU putative oxidase, FAD-binding subunit 103 usg putative aspartate-semialdehyde dehydrogenase, NAD(P)-binding (2nd module) 104 der putative GTP-binding factor (1st module) 105 yqfA putative transmembrane protein 106 yncC putative transcriptional regulator (GntR familiy) 107 bo373 Putative transposase-related protein 108 phoH PhoB-dependent, ATP-binding pho regulon component (2nd module) 

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