UC Berkeley UC Berkeley Electronic Theses and Dissertations Title An evaluation of diarrheagenic Escherichia coli survival after ingestion by Tetrahymena sp. and Helicobacter pylori's fate after ingestion by Tetrahymena sp. and Acanthamoeba polyphaga Permalink https://escholarship.org/uc/item/3gs815mp Author Smith, Charlotte Dery Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California An evaluation of diarrheagenic Escherichia coli survival after ingestion by Tetrahymena sp. and Helicobacter pylori’s fate after ingestion by Tetrahymena sp. and Acanthamoeba polyphaga by Charlotte Dery Smith A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Environmental Health Sciences in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Robert C. Spear, Chair Professor Michael N. Bates Professor Lee W. Riley Professor Sharon G. Berk Spring 2012 An evaluation of diarrheagenic Escherichia coli survival after ingestion by Tetrahymena sp. and Helicobacter pylori’s fate after ingestion by Tetrahymena sp. and Acanthamoeba polyphaga Copyright 2012 Charlotte Dery Smith Abstract An evaluation of diarrheagenic Escherichia coli survival after ingestion by Tetrahymena sp. and Helicobacter pylori’s fate after ingestion by Tetrahymena sp. and Acanthamoeba polyphaga by Charlotte Dery Smith Doctor of Philosophy in Environmental Health Sciences University of California, Berkeley Professor Robert C. Spear, Chair Diarrheagenic pathotypes of Escherichia coli are responsible for a substantial number of childhood deaths each year, and Helicobacter pylori causes gastric diseases in both children and adults worldwide. Free-living protozoa have been implicated in the survival and transport of pathogens in the environment, but the relationship between either non-Shiga toxin-producing E. coli or H. pylori and free-living ciliated protozoa has not been characterized. Likewise, there is a scarcity of research regarding the ability of these pathogenic bacteria to evade destruction during phagocytosis by amoebae. A literature review revealed two articles demonstrating survival of E. coli O157:H7 and or environmental isolate of E. coli in Acanthamoeba and one article reporting survival of H. pylori in Acanthamoeba. There have been several reports of E. coli O157:H7 or genetically modified laboratory strains surviving digestion by Tetrahymena, and no investigations on the resistance of H. pylori to digestion by this aquatic ciliate. Six diarrheagenic serotypes of E. coli and an isolate of H. pylori were evaluated for their susceptibility to digestion by Tetrahymena. Tetrahymena strain MB125, were fed E. coli or H. pylori and the ciliate’s egested products examined for viable pathogens. Viability was assessed by the BacLight™ LIVE/DEAD™ assay, by a cell elongation method, and by colony counts. An H. pylori strain expressing the green fluorescent protein, an ATCC strain originating from a clinical isolate, and a fresh clinical isolate of H. pylori were fed to Acanthamoeba polyphaga. Viability of H. pylori in Acanthamoeba was assessed with the BacLight™ LIVE/DEAD™ assay, fluorescent in situ hybridization, and quantitation of amplified gene products over time as determined by a real time polymerase chain reaction test. All six pathogenic E. coli serotypes survived digestion by Tetrahymena and emerged as viable cells in fecal pellets, whereas H. pylori was digested. Growth of E. coli on agar plates indicated that the bacteria were able to replicate after passage through the ciliate. Transmission electron micrographs of E. coli cells as intact rods versus degraded H. pylori cells corroborated these results. Scanning electron microscopy revealed a net-like matrix around intact E. coli cells in fecal pellets. Helicobacter pylori did not survive phagocytosis by Acanthamoeba. These results suggest a possible role for Tetrahymena and its egested fecal pellets in the dissemination of diarrheagenic E. coli in the environment. This bacterial-protozoan association may increase opportunities for transmission of these bacterial pathogens to mammalian hosts, including humans. 1 Dedication This dissertation is dedicated to Jim Smith. My husband, my best friend, my sole-mate, my whole world. and To my parents, Evon and Julian Dery, who during their lives said so many wise things to me like: “Get all the education you can, nobody can ever take that away from you” and “nobody ever got into trouble by listening too much”. i Contents Chapter 1 1 Introduction Chapter 2 21 Helicobacter pylori phagocytosis by Acanthamoeba polyphaga demonstrated by microscopy, fluorescent in situ hybridization, and quantitative polymerization chain reaction tests Chapter 3 33 Survival characteristics of diarrheagenic Escherichia coli pathotypes and Helicobacter pylori during passage through the free-living ciliate, Tetrahymena sp. Chapter 4 47 Enumeration of E. coli when associated with the free-living ciliate, Tetrahymena sp.: Comparison of Colilert-18® and MacConkey agar media Chapter 5 55 Conclusions and future research ii Acknowledgements I am grateful to my dissertation committee, Professor Robert C. Spear, Professor Lee W. Riley, Professor Michael N. Bates, and Professor Sharon G. Berk for their unwavering support throughout this journey. I thank Dr. Maria Brandl, USDA, not only for the gift of Tetrahymena MB125, and for her enthusiastic encouragement especially during the writing of a shared manuscript. Many thanks to Drs. Nickolas Ashbolt, USEPA and Graciela Ramirez-Toro, Inter American University of Puerto Rico who helped launch the dissertation project. Thanks also to my colleagues during my year at the EPA in Cincinnati who helped me in so many ways: Stacy Pfaller, Maura Donohue, Gretchen Sullivan, John Hall, Eric and Leah Villegas, Patricia Klonicki and Keya Sen, who shared her H. pylori method with me. All of the professors at Berkeley were wonderful, but some are very special to me especially Maureen Lahiff for her instruction, patience and encouragement even in the years after I took her classes, Warren Winkelstein my first professor at Berkeley who inspired me with his strength and commitment to Public Health. To George Sensabaugh, Jon Schwartzberg, Jack Colford, Steve Selvin, Nick Jewel: thank you for your wisdom and kindness. Many thanks to Holly Arron, Reena Zapuri and Ken McDonald at the UCB Core Microscopy Centers who taught me the skills to express the artistic side of my project. I appreciate the excellent laboratory assistance from Oichee Bose and Joanne Dai. To the people whose friendship sustained me while I took a sabbatical from my life to venture back to academia: Marjorie Shovlin, Tom Cleveland, Marty Laporte, Linda and Phil Meyers, Maria Jose Figueras, Amador, Rey and Zoraida Goodrich, Juliana Matos de Arruda and Troy Shahoumian: Thank you so very much! Ron and Isabel Hunsinger and Linda and Walter Grayman shared so many good times, and were there for me during the hard times. You are like family to me and there are no words to express my gratitude sufficiently. To my far away and forever friends Kim Brown and Sue Borel, no matter where life takes us, our hearts will always be close. Green fluorescent protein – Helicobacter pylori developed by Andrew Wright (Tufts University) was a gift from Kathryn Eaton (University of Michigan). The H. pylori clinical isolate was a gift from Richard Peek (Vanderbilt University). iii Chapter 1 Introduction The goal of this dissertation research is to elucidate the association between two enteric pathogens and two types of free-living protozoa. The enteric pathogens Helicobacter pylori and diarrheagenic Escherichia coli contribute to the global burden of disease resulting in both serious illness and death. However, their ecology is not well defined especially in terms of interactions with other microbes. Microbial ecologists have begun to define relationships among a variety of pathogenic bacteria and two ubiquitous aquatic protozoan genera, the amoeba, Acanthamoeba, and the ciliate, Tetrahymena, leading to important public health implications. Certain characteristics of these protozoa may support proliferation of enteric bacterial pathogens in water systems, either by sheltering them from disinfectants1 or creating a disinfectant demand so that disinfectants have diminished capacity to kill the bacteria. The latter would be a concern if the two phyla (protozoa and bacteria) are found within a biofilm or agglomeration of microbial cells (e.g., floc in a wastewater plant). Acanthamoeba cysts are resistant to chlorine at more than 10 - 50 times higher concentrations than those typically used to disinfect water supplies. Tetrahymena exudes sticky substances from intracellular organelles called mucocysts and egest fecal pellets that contribute to floc formation.2-5 This chapter introduces the organisms as a background for the following chapters that describe the ability of Helicobacter pylori and diarrheagenic Escherichia coli to survive predation by Acanthamoeba and Tetrahymena. Acanthamoeba Acanthamoeba is a genus in the family Acanthmoebidae in the phylum Amoebozoa, characterized by movement facilitated by cytoplasmic flow. Amoebae are recognized by distinct projections called pseudopodia that are involved in locomotion as well as capture of prey. Free- living protozoa, such as Acanthamoeba, rely on bacterial prey for their survival. Digestion occurs by acidification