DOCSLIB.ORG

Genetic and Phenotypic Characterization of Livestock Associated Methicillin Resistant Staphylococcus Aureus Sequence Type (ST) 5

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genetic and Phenotypic Characterization of Livestock Associated Methicillin Resistant Staphylococcus Aureus Sequence Type (ST) 5 Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 Genetic and phenotypic characterization of livestock associated methicillin resistant Staphylococcus aureus sequence type (ST) 5 in comparison with clinical ST5 isolates from humans Samantha Jean Hau Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Microbiology Commons Recommended Citation Hau, Samantha Jean, "Genetic and phenotypic characterization of livestock associated methicillin resistant Staphylococcus aureus sequence type (ST) 5 in comparison with clinical ST5 isolates from humans" (2017). Graduate Theses and Dissertations. 16143. https://lib.dr.iastate.edu/etd/16143 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Genetic and phenotypic characterization of livestock associated methicillin resistant Staphylococcus aureus sequence type (ST) 5 in comparison with clinical ST5 isolates from humans by Samantha Jean Hau A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Veterinary Microbiology Program of Study Committee: Qijing Zhang, Co-Major Professor Timothy S. Frana, Co-Major Professor Steve A. Carlson Ganwu Li Tracy L. Nicholson The student author and the program of study committee are solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2017 Copyright © Samantha Jean Hau, 2017. All rights reserved. ii DEDICATION For L.B. Your enthusiasm and sense of adventure make life more interesting. iii TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................... viii LIST OF TABLES ........................................................................................................... ix NOMENCLATURE ...........................................................................................................x ACKNOWLEDGMENTS ............................................................................................. xiii ABSTRACT .................................................................................................................... xiv CHAPTER 1. GENERAL INTRODUCTION AND LITERATURE REVIEW .........1 General Biology of Staphylococcus aureus ...................................................................1 S. aureus Classification and Genetics ...........................................................................2 S. aureus Colonization and Disease ..............................................................................5 S. aureus Virulence Factors and Pathogenesis ............................................................7 Adhesion to host tissues ...............................................................................................7 Biofilm formation ......................................................................................................10 Nutrient acquisition ....................................................................................................10 Invasion and tissue destruction ..................................................................................10 Toxin mediated disease ..............................................................................................11 Immune evasion .........................................................................................................14 Drug Resistance in S. aureus .......................................................................................16 Methicillin Resistance ..................................................................................................23 MRSA Epidemiology ...................................................................................................27 LA-MRSA Characteristics ..........................................................................................32 Aims and Significance of Research ............................................................................36 Organization of Dissertation .......................................................................................38 CHAPTER 2. SINGLE NUCLEOTIDE POLYMORPHISM ANALYSIS INDICATES GENETIC DISTINCTION AND REDUCED DIVERSITY OF AGRICULTURAL METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) ST5 ISOLATES AS COMPARED TO CLINICAL MRSA ST5 ISOLATES ................................................................................................................39 Abstract .........................................................................................................................40 Introduction ..................................................................................................................40 Methods .........................................................................................................................43 Isolate Acquisition .....................................................................................................43 Whole Genome Sequencing .......................................................................................43 iv Core Genome SNP Discovery ...................................................................................44 Mobile genetic element analysis ................................................................................46 Results ...........................................................................................................................47 Isolate provenance and sequence information ...........................................................47 Single nucleotide polymorphism typing ....................................................................47 Phylogenetic analysis .................................................................................................49 Mobile genetic element analysis ................................................................................52 Discussion......................................................................................................................54 Acknowledgements ......................................................................................................56 CHAPTER 3. COMPARATIVE PREVALENCE OF IMMUNE EVASION COMPLEX GENES ASSOCIATED WITH BETA-HEMOLYSIN CONVERTING BACTERIOPHAGES IN MRSA ST5 ISOLATES FROM SWINE, SWINE FACILITIES, HUMANS WITH SWINE CONTACT, AND HUMANS WITH NO SWINE CONTACT ...................................................................57 Abstract .........................................................................................................................57 Introduction ..................................................................................................................59 Methods .........................................................................................................................63 Strain acquisition .......................................................................................................63 DNA isolation ............................................................................................................63 PCR reactions.............................................................................................................64 Southern blotting ........................................................................................................66 Phage typing...............................................................................................................66 Statistical analysis ......................................................................................................67 Results ...........................................................................................................................67 IEC gene prevalence ..................................................................................................67 β-hemolysin converting bacteriophage prevalence via integrase gene screening .....69 β-hemolysin gene analysis .........................................................................................70 Phage typing...............................................................................................................72 Discussion......................................................................................................................73 Acknowledgments ........................................................................................................77 CHAPTER 4. ZINC RESISTANCE WITHIN SWINE-ASSOCIATED METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATES IN THE UNITED STATES IS ASSOCIATED WITH MULTILOCUS SEQUENCE TYPE LINEAGE .............................................................................................................78 Abstract .........................................................................................................................78 Importance....................................................................................................................79 v Introduction ..................................................................................................................80
Load more

Recommended publications
  • Piggybac Transposase Tools for Genome Engineering.Pdf
    piggyBac transposase tools for genome engineering PNAS PLUS Xianghong Lia,b, Erin R. Burnightc, Ashley L. Cooneyd, Nirav Malanie, Troy Bradye, Jeffry D. Sanderf,g, Janice Staberd, Sarah J. Wheelanh, J. Keith Joungf,g, Paul B. McCray, Jr.c,d, Frederic D. Bushmane, Patrick L. Sinnd,1, and Nancy L. Craiga,b,1 aHoward Hughes Medical Institute and bDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cGenetics Program, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242; dDepartment of Pediatrics, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242; eDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; fMolecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02129; gDepartment of Pathology, Harvard Medical School, Boston, MA 02115; and hDivision of Biostatistics and Bioinformatics, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved May 1, 2013 (received for review December 30, 2012) The transposon piggyBac is being used increasingly for genetic stud- site-directed mutagenesis of the PB catalytic domain and isolated − ies. Here, we describe modified versions of piggyBac transposase an excision competent/integration defective (Exc+Int ) PB. We − that have potentially wide-ranging applications, such as reversible also find that introduction of the Exc+Int mutations into a hy- − transgenesis and modified targeting of insertions. piggyBac is dis- peractive version of PB (5, 8, 15) yields an Exc+Int transposase tinguished by its ability to excise precisely, restoring the donor site whose excision frequency is five- to six-fold higher than that of to its pretransposon state.
    [Show full text]
  • Comparative Genomics of Staphylococcus Reveals Determinants of Speciation and Diversification of Antimicrobial Defense
    1 Comparative Genomics of Staphylococcus Reveals Determinants of 2 Speciation and Diversification of Antimicrobial Defense. 3 4 5 Rosanna Coates-Brown1§, Josephine Moran1, Pisut Pongchaikul1¶, Alistair Darby1 and 6 Malcolm J. Horsburgh1* 7 8 9 10 11 12 1Institute of Integrative Biology, University of Liverpool, Liverpool, Merseyside, United 13 Kingdom. 14 15 § Present address: Genomic Diagnostic Laboratory, St Mary’s Hospital, Oxford Road, 16 Manchester, UK 17 ¶Present address: Faculty of Medicine Ramathibodi Hospital, Mahidol University, 270 18 Rama IV Road, Ratchathewi, Bangkok, 10400, Thailand 19 20 21 * Corresponding author: Institute of Integrative Biology, University of Liverpool, 22 Liverpool, L69 7ZB, United Kingdom. 23 Email: [email protected] 24 Tel: +44 1517954569 25 Fax +44 1517954410 26 Abstract 27 The bacterial genus Staphylococcus comprises diverse species with most being described 28 as colonizers of human and animal skin. A relational analysis of features that 29 discriminate its species and contribute to niche adaptation and survival remains to be fully 30 described. In this study, an interspecies, whole-genome comparative analysis of 21 31 Staphylococcus species was performed based on their orthologues. Three well-defined 32 multi-species groups were identified: group A (including aureus/epidermidis); group B 33 (including saprophyticus/xylosus) and group C (including pseudintermedius/delphini). 34 The machine learning algorithm Random Forest was applied to prioritise orthologues that 35 drive formation of the Staphylococcus species groups A-C. Orthologues driving 36 staphylococcal intrageneric diversity comprised regulatory, metabolic and antimicrobial 37 resistance proteins. Notably, the BraSR (NsaRS) two-component system (TCS) and its 38 associated BraDE transporters that regulate antimicrobial resistance showed limited 39 Distribution in the genus and their presence was most closely associated with a subset of 40 Staphylococcus species dominated by those that colonise human skin.
    [Show full text]
  • Staphylococcus Edaphicus Sp
    EVOLUTIONARY AND GENOMIC MICROBIOLOGY crossm Staphylococcus edaphicus sp. nov., Isolated in Antarctica, Harbors the mecC Gene and Genomic Islands with a Suspected Role in Adaptation to Extreme Environments Roman Pantu˚cˇek,a Ivo Sedlácˇek,b Adéla Indráková,a Veronika Vrbovská,a,b Ivana Mašlanˇová,a Vojteˇch Kovarˇovic,a Pavel Švec,b Stanislava Králová,b Lucie Krištofová,b Jana Kekláková,c Petr Petráš,c Jirˇí Doškarˇa aDivision of Genetics and Molecular Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic bCzech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic cReference Laboratory for Staphylococci, National Institute of Public Health, Prague, Czech Republic ABSTRACT Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staph- ylococcus saprophyticus. However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of Ͻ85% and inferred DNA-DNA hybridization of Ͻ30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeep- ing genes, rpoB, hsp60, tuf, dnaJ, gap, and sod. Matrix-assisted laser desorption ion- ization–time of flight mass spectrometry (MALDI-TOF MS) and key biochemical charac- teristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S.
    [Show full text]
  • Frequency and Characterization of Antimicrobial Resistance and Virulence Genes of Coagulase-Negative Staphylococci from Wild Birds in Spain
    microorganisms Article Frequency and Characterization of Antimicrobial Resistance and Virulence Genes of Coagulase-Negative Staphylococci from Wild Birds in Spain. Detection of tst-Carrying S. sciuri Isolates Laura Ruiz-Ripa 1, Paula Gómez 1, Carla Andrea Alonso 2 , María Cruz Camacho 3, Yolanda Ramiro 3, Javier de la Puente 4,5, Rosa Fernández-Fernández 1, Miguel Ángel Quevedo 6, Juan Manuel Blanco 7, Gerardo Báguena 8, Myriam Zarazaga 1, Ursula Höfle 3 and Carmen Torres 1,* 1 Área de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain; [email protected] (L.R.-R.); [email protected] (P.G.); [email protected] (R.F.-F.); [email protected] (M.Z.) 2 Servicio de Microbiología, Hospital San Pedro, 26006 Logroño, Spain; [email protected] 3 Grupo SaBio, Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), 13005 Ciudad Real, Spain; [email protected] (M.C.C.); [email protected] (Y.R.); ursula.hofl[email protected] (U.H.) 4 SEO/BirdLife, Citizen Science Unit, 28053 Madrid, Spain; [email protected] 5 Parque Nacional de la Sierra de Guadarrama, Centro de Investigación, Seguimiento y Evaluación, 28740 Rascafría, Spain 6 Zoobotánico Jerez, 11408 Jerez de la Frontera, Spain; [email protected] 7 Aquila Foundation, 45567 Oropesa, Spain; [email protected] 8 Fundación para la Conservación del Quebrantahuesos, 50001 Zaragoza, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-941299750 Received: 4 August 2020; Accepted: 26 August 2020; Published: 29 August 2020 Abstract: The objective of this study was to determine the prevalence and diversity of coagulase-negative staphylococci (CoNS) species from wild birds in Spain, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content.
    [Show full text]
  • Bacteria Associated with Larvae and Adults of the Asian Longhorned Beetle (Coleoptera: Cerambycidae)1
    Bacteria Associated with Larvae and Adults of the Asian Longhorned Beetle (Coleoptera: Cerambycidae)1 John D. Podgwaite2, Vincent D' Amico3, Roger T. Zerillo, and Heidi Schoenfeldt USDA Forest Service, Northern Research Station, Hamden CT 06514 USA J. Entomol. Sci. 48(2): 128·138 (April2013) Abstract Bacteria representing several genera were isolated from integument and alimentary tracts of live Asian longhorned beetle, Anaplophora glabripennis (Motschulsky), larvae and adults. Insects examined were from infested tree branches collected from sites in New York and Illinois. Staphylococcus sciuri (Kloos) was the most common isolate associated with adults, from 13 of 19 examined, whereas members of the Enterobacteriaceae dominated the isolations from larvae. Leclercia adecarboxylata (Leclerc), a putative pathogen of Colorado potato beetle, Leptinotarsa decemlineata (Say), was found in 12 of 371arvae examined. Several opportunistic human pathogens, including S. xylosus (Schleifer and Kloos), S. intermedius (Hajek), S. hominis (Kloos and Schleifer), Pantoea agglomerans (Ewing and Fife), Serratia proteamaculans (Paine and Stansfield) and Klebsiella oxytoca (Fiugge) also were isolated from both larvae and adults. One isolate, found in 1 adult and several larvae, was identified as Tsukamurella inchonensis (Yassin) also an opportunistic human pathogen and possibly of Korean origin .. We have no evi­ dence that any of the microorganisms isolated are pathogenic for the Asian longhorned beetle. Key Words Asian longhorned beetle, Anaplophora glabripennis, bacteria The Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) a pest native to China and Korea, often has been found associated with wood- packing ma­ terial arriving in ports of entry to the United States. The pest has many hardwood hosts, particularly maples (Acer spp.), and currently is established in isolated popula­ tions in at least 3 states- New York, NJ and Massachusetts (USDA-APHIS 201 0).
    [Show full text]
  • A Homologous Recombination-Based Method of Genetic Engineering
    PROTOCOL Recombineering: a homologous recombination-based method of genetic engineering Shyam K Sharan1, Lynn C Thomason2, Sergey G Kuznetsov1 & Donald L Court3 1Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. 2SAIC-Frederick Inc., Gene Regulation and Chromosome Biology Laboratory, Basic Research Program, Frederick, Maryland, 21702, USA. 3Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. Correspondence should be addressed to S.K.S. ([email protected]) or D.L.C. ([email protected]). Published online 29 January 2009; doi:10.1038/nprot.2008.227 Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-stranded linear DNA substrates, natureprotocols so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene / m tags can be inserted and regions of bacterial artificial chromosomes or the E. coli genome can be subcloned by gene retrieval using o c . recombineering. Most of these constructs can be made within about 1 week’s time. e r u t a n . INTRODUCTION w w Recombineering is an in vivo method of genetic engineering used This protocol will emphasize modification of bacterial artificial w / / 1–5 : primarily in Escherichia coli that uses short 50 base homologies .
    [Show full text]
  • Table of Contents
    An Investigation of Staphylococcus aureus and Related Species From Flood Affected and Other Environmental Sources A Thesis in Molecular Microbiology by Nadeesha Samanmalee Jayasundara BSc (Environmental Conservation & Management) School of Biomedical Science, Institute of Health & Biomedical Innovation Queensland University of Technology Brisbane, Australia Thesis submitted to Queensland University of Technology in fulfilment of the requirements for the degree of Masters of Applied Science (Research) May 2014 2 Abstract The genus Staphylococcus consists of 45 species and is widely distributed across environments such as skin and mucous membranes of humans and animals, as well as in soil, water and air. S. aureus and S. epidermidis are the most commonly associated species with human infections. Hence, most studies have focused on clinical and clinically sourced staphylococci. In addition, S. haemoliticus, S. intermidius, S. delphini, and S. saprophiticus are also considered potentially pathogenic members of the genus. Although staphylococci are distributed in various environments, there have been very few studies examining residential air as a reservoir of clinically significant pathogens, particularly Staphylococcus species. As a result, airborne transmission of staphylococci, and associated health risks, remains unclear. This study included not only residential air but also air samples from flood affected houses. Flood water can be considered as a potential carrier of pathogenic bacteria, because flood water can be affected by residential septic systems, municipal sanitary sewer systems, hospital waste, agricultural lands/operations and wastewater treatment plants. Even after the flood waters recede, microorganisms that are transported in water can remain in soil, in or on plant materials and on numerous other surfaces. Therefore, there is a great concern for use of previously flooded indoor and outdoor areas.
    [Show full text]
  • An Optimized Preparation Method for Long Ssdna Donors to Facilitate Quick Knock-In Mouse Generation
    cells Article An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation Yukiko U. Inoue 1,* , Yuki Morimoto 1, Mayumi Yamada 2, Ryosuke Kaneko 3, Kazumi Shimaoka 1, Shinji Oki 4, Mayuko Hotta 1, Junko Asami 1, Eriko Koike 1, Kei Hori 1, Mikio Hoshino 1, Itaru Imayoshi 2,5 and Takayoshi Inoue 1,* 1 Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan; [email protected] (Y.M.); [email protected] (K.S.); [email protected] (M.H.); [email protected] (J.A.); [email protected] (E.K.); [email protected] (K.H.); [email protected] (M.H.) 2 Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; [email protected] (M.Y.); [email protected] (I.I.) 3 KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; [email protected] 4 Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan; [email protected] 5 Department of Deconstruction of Stem Cells, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan * Correspondence: [email protected] (Y.U.I.); [email protected] (T.I.) Abstract: Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles Citation: Inoue, Y.U.; Morimoto, Y.; in investigating various molecular functions in vivo.
    [Show full text]
  • Genetic Diversity of Methicillin Resistant Staphylococcus Aureus Strains in the Pretoria
    Genetic diversity of methicillin resistant Staphylococcus aureus strains in the Pretoria region in South Africa ADEOLA MUJIDAT SALAWU Genetic diversity of methicillin resistant Staphylococcus aureus strains in the Pretoria region in South Africa by ADEOLA MUJIDAT SALAWU Submitted in partial fulfilment of the requirements for the degree MAGISTER SCIENTIAE MSc (Medical Microbiology) Department of Medical Microbiology Faculty of Health Sciences University of Pretoria Gauteng South Africa October 2013 Declaration I, the undersigned, declare that the dissertation hereby submitted to the University of Pretoria for the degree MSc (Medical Microbiology) and the work contained herein is my original work and has not previously, in its entirety or in part, been submitted to any university for a degree. I further declare that all sources cited are acknowledged by means of a list of references. Signed_________________this_________________day of_________________2014 Spending time with GOD is the key to our strength and success in all areas of life. Be sure that you never try to work GOD into your schedule, but always work your schedule around HIM Joyce Meyer Dedication To my dear husband (Babatunde Rotimi ): Thank you for your support, love and understanding ACKNOWLEDGEMENTS Firstly: I would like to extend my greatest gratitude to the almighty GOD, the creator of heaven and earth. My GOD, I will forever be grateful to You Secondly: I would like to sincerely thank the following individuals: Prof MM Ehlers (supervisor), Department of Medical Microbiology, University of Pretoria/NHLS, for her humility, endless motivation, patience, support and professional supervision in the successful completion of this research project. Prof, GOD bless you Dr MM Kock (co-supervisor), Department of Medical Microbiology, University of Pretoria/NHLS, for her guidance, understanding, support and molecular biology expertise regarding this research project.
    [Show full text]
  • Transposon Based Mutagenesis and Mapping of Transposon Insertion Sites Within the Ehrlichia Chaffeensis Genome Using Semi Random Two-Step Pcr
    TRANSPOSON BASED MUTAGENESIS AND MAPPING OF TRANSPOSON INSERTION SITES WITHIN THE EHRLICHIA CHAFFEENSIS GENOME USING SEMI RANDOM TWO-STEP PCR by VIJAYA VARMA INDUKURI B.S., ANDHRA UNIVERSITY, India, 2008 A THESIS Submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2013 Approved by: Major Professor Roman Reddy Ganta Copyright VIJAYA VARMA INDUKURI 2013 Abstract Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment.
    [Show full text]
  • The Evolutionary History of Chromosomal Super-Integrons Provides an Ancestry for Multiresistant Integrons
    The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons Dean A. Rowe-Magnus*, Anne-Marie Guerout*, Pascaline Ploncard*, Broderick Dychinco†, Julian Davies†, and Didier Mazel*‡ *Unite´de Programmation Mole´culaire et Toxicologie Ge´ne´ tique, Centre National de la Recherche Scientifique Unite´de Recherche Associe´e 1444, De´partement des Biotechnologies, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris, France; and †Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3 Communicated by Allan Campbell, Stanford University, Stanford, CA, November 2, 2000 (received for review August 1, 2000) Integrons are genetic elements that acquire and exchange exog- located downstream of a resident promoter internal to the intI enous DNA, known as gene cassettes, by a site-specific recombi- gene, drives expression of the encoded proteins (13). Most of the nation mechanism. Characterized gene cassettes consist of a target attC sites of the integron cassettes identified to date are unique. recombination sequence (attC site) usually associated with a single Their length and sequence vary considerably (from 57 to 141 bp) open reading frame coding for an antibiotic resistance determi- and their similarities are primarily restricted to their boundaries, nant. The affiliation of multiresistant integrons (MRIs), which which correspond to the inverse core site (RYYYAAC) and the contain various combinations of antibiotic resistance gene cas- core site [G2TTRRRY; 2, recombination point (11, 14)]. settes, with transferable elements underlies the rapid evolution of More than 60 different antibiotic-resistance genes, covering multidrug resistance among diverse Gram-negative bacteria. Yet most antimicrobials presently in use, have been characterized in the origin of MRIs remains unknown.
    [Show full text]
  • Electroporation-Mediated and EBV LMP1-Regulated Gene Therapy in A
    Cancer Gene Therapy (2003) 10, 626–636 r 2003 Nature Publishing Group All rights reserved 0929-1903/03 $25.00 www.nature.com/cgt Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model Yu-hua Hsieh,1 Chang-jer Wu,2 Kai-ping Chow,3 Chia-lung Tsai,1 and Yu-sun Chang3 1Institute of Microbiology and Immunology, National Yang-Ming University, Shih-pai, Taipei 112, Republic of China; 2Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Republic of China; and 3Graduate Institute of Basic Medical Sciences, Chang-Gung University, Kwei-shan, Taoyuan 333, Taiwan, Republic of China. Latent membrane protein 1 (LMP1) is an Epstein–Barr virus (EBV)-encoded oncogene expressed in EBV-associated nasopharyngeal carcinoma (NPC). Previous studies indicate that a strategy combining LMP1-mediated NF-kB activation and the HSV thymidine kinase/Ganciclovir (HSVtk/GCV) prodrug system leads to regression of tumor growth in nude mice. To improve the efficacy of this strategy in immunocompetent hosts, we developed a therapeutic cassette, p6kB-EDL1E-tk, containing six copies of the NF-kB binding motif and an epithelial-specific EBV promoter, ED-L1E. The cassette was tested in a murine CT-26 carcinoma model in syngenic Balb/c mice. Coinjection of an LMP1-expressing vector and p6kB-EDL1E-tk by in vivo electroporation in mouse muscle revealed at least two-fold higher TK enzymatic activity than that of previously tested pLTR-tk. Furthermore, growth was attenuated in a group of mice containing LMP1-positive tumors that were intratumorally injected with the p6kB-EDL1E-tk cassette and GCV via in vivo electroporation, but not in mice treated with p6kB-EDL1E-tk or GCV alone.
    [Show full text]