DOCSLIB.ORG

Fusobacterium Pathogenesis Using Molecular and Genomic Methods to Inform Vaccine Design

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Fusobacterium Pathogenesis Using Molecular and Genomic Methods to Inform Vaccine Design This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Brewer, Matt L Title: Investigating Fusobacterium pathogenesis using molecular and genomic methods to inform vaccine design General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Investigating Fusobacterium pathogenesis using molecular and genomic methods to inform vaccine design Matthew L. Brewer A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences School of Biochemistry, September 2018 Word Count: 38419 Abstract Abstract Cellular adhesion is crucial in the pathogenesis of many bacterial species, where receptor- ligand interactions mediate host colonisation and/or invasion leading to host pathologies. Fusobacterium nucleatum (Fn) is a bacterial species gaining greater interest due to recent links with a variety of diseases, such as colorectal cancer. Fn is known to harbour a vast array of adhesive proteins (adhesins), however only a few have been studied in depth, such as FadA. This study aims to characterise a new previously unstudied subset of trimeric autotransporter adhesins (TAAs) found within Fn, responsible for binding the human receptor CEACAM1. These proteins were given the name CEACAM-binding proteins of Fusobacterium (CbpF). To examine the distribution of these receptors among Fusobacterium spp., we screened and sequenced a library of clinical isolates. From sequencing these strains, we identified two novel species of Fusobacterium (F. oralis sp. nov. and F. ovarium sp. nov.), both of which harboured CbpF. While performing the taxonomic analyses on the new strains we addressed the conflicting nomenclature and phylogenetic boundaries with respect to the genus. By utilising computational methods, we could confidently delineate species and show how the genus should be organised to better reflect the genomic differences and similarities between strains. Through screening two different types of CbpF from different species we confirmed the ability of both classes to bind CEACAM1 through proteomic- and cellular adhesion-based assays as well as showing that both classes of protein were capable of binding to CEA (CEACAM5), but not to other CEACAM variants examined. This highlighted the highly specific nature of these proteins, which was explored further by examining point mutants of CEACAM1, of which few showed any significant adhesion. As well as examining CbpF, we briefly looked at two other TAAs from Fn: FN0471 and FN0735; the former of which could bind indiscriminately to HeLa cells, thus indicating another important adhesin yet to be fully characterised. Structural analysis of CbpFs highlighted a gap in the literature with respect to TAA motifs and topologies, where no known structures showed significant homology to large portions of the proteins particularly in a region predicted to be occupied with a coiled-coil motif. X-ray crystallography, SAXS and CD were used to infer structural features of CbpF, however an atomic resolution structure could not be accurately produced from a protein crystal X-ray diffraction dataset. The work conducted here lays the foundation for additional studies into TAAs from Fusobacterium highlighting the requirement for increased detail on how these proteins contribute to pathogenesis and whether these proteins could be used as potential future vaccine candidates. i Acknowledgements Acknowledgements I would like to thank my supervisors, Dr Darryl Hill and Prof Leo Brady for providing me the opportunity to do this PhD as well as their unwavering support throughout its duration. Additionally, I would like to thank the SWBio DTP for accepting me and putting together a great doctoral training course. I also have to thank my progression panel, Ross and Paul, for providing additional support and helping to keep me on track throughout. Secondly, I have to say a huge thank you to all the many people over the last four years that have taught me the vast array of techniques I used in this work. Firstly, to Clio for helping me massively in my early stages in the lab, as well as providing me with the wisdom of a newbie post-doc. Other members of the Hill and Brady groups that deserve my appreciation are Ahmad and Storm for showing me the ways of protein purification and to Darryl and Nibras with tissue culture and cell imaging etc. Honourable mentions go to Nick, Ash, Johnny, Sesh and various DLS staff for all their help with SAXS, CD, MD and X-ray crystallography. In addition to the practical and academic support, I would like to thank our wonderful C-floor tech team, including Doug, Dave, Andy, Bill and Anne for all the general lab equipment support as well as making some of the less desirable buffers and media! Especially Anne for short notice media orders. I also want to thank my office pals including Henry, Alex and various other people, including the Finn group, that have had to put up with my (and Henry’s) antics over these past years. Without the relaxed working environment, I don’t think I would have stayed sane over the course of my PhD. Lastly, I would like to thank Bronwyn, my family and various other friends for being there to listen to me waffle on endlessly about a topic they clearly have no interest in and to give me a chance to not have to think about science. ii Author’s Declaration Author’s Declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. Signed: .................................... Date: ........................................ iii Contents Contents Abstract i Acknowledgements ii Author’s Declaration iii Contents iv List of Figures x List of Tables xiii Acronyms and Abbreviations xiv Chapter 1: Introduction 1 1.1 The Fusobacterium genus 1 1.1.1 Biophysical and Biochemical Characteristics 1 1.1.2 Taxonomy 2 1.1.3 Health and Disease 4 1.1.3.1 Periodontal Disease 4 1.1.3.2 Colorectal cancer 6 1.1.3.3 Preterm births 7 1.1.3.4 Lemierre’s Syndrome 7 1.1.3.5 Footrot 8 1.1.3.6 Other Diseases 9 1.1.3.7 Treatment 9 1.1.4 Virulence Factors 9 1.1.4.1 Adhesive Proteins 10 1.2 Trimeric Autotransporter Adhesins 11 iv Contents 1.2.1 Structure and Assembly 11 1.2.2 Specialised folds 13 1.2.3 Receptor binding 18 1.3 CEACAMs 19 1.3.1 Functions 19 1.3.2 Structural Properties 22 1.3.3 Pathogen Interplay and Disease 26 1.4 Fusobacterium Vaccine Antigen Targets 27 1.5 Aims 28 Chapter 2: Materials and Methods 28 2.1 Bacterial strains and growth conditions 28 2.2 Eukaryotic strains and growth conditions 30 2.3 Gene Cloning 31 2.3.1 Creating a Plasmid for Producing Soluble Recombinant Protein 31 2.3.2 Creating a Plasmid for Surface Expression of Protein 33 2.3.3 Site-directed Mutagenesis 33 2.3.4 Plasmids and Primers 35 2.4 Bacterial Protein Expression and Purification 39 2.4.1 Small-scale Expression and Native Purification 39 2.4.2 Large-scale Expression and Native Purification 40 2.4.3 Large-scale Expression and Denaturing Purification 41 2.4.4 Surface Expression 41 2.5 Fusobacterium Lysate Preparation 41 v Contents 2.6 Human CEACAM IgG1-FC fusion protein production 42 2.6.1 DNA Preparation 42 2.6.2 Large Scale Transient Transfection 42 2.6.3 Small-scale Transfections and Purification 43 2.7 Western blots, Immunodot blots and ELISAs 43 2.7.1 Quantitative ELISA 44 2.8 Adhesion assays 45 2.8.1 Eukaryotic cell preparation 45 2.8.2 Inhibitor preparation 45 2.8.3 Bacterial preparation 45 2.8.4 Cell fixation 46 2.8.5 Cell staining 46 2.8.6 Cell imaging 47 2.9 Crystallography 47 2.9.1 Crystal Looping and Data Collection 48 2.10 Small-angle X-ray Scattering 48 2.11 Molecular Dynamics 49 2.12 Whole Genome Sequencing 49 2.13 Phylogenetics 50 2.13.1 Genome and Proteome Mining 50 2.13.2 Average Nucleotide Identity 50 2.13.3 Maximal Unique Matches and MUMi 50 2.13.4 Pan-Locus Sequence Analysis 51 vi Contents 2.13.5 Phylogenetic Trees 52 2.13.6 Hive Plots 53 2.14 Statistics 53 Chapter 3: A comprehensive phylogenetic analysis of the Fusobacterium genus 54 3.1 Introduction 54 3.2 Whole-Genome Sequencing of Clinical Strains 56 3.3 Method Comparison 59 3.3.1 Pre-screening for Non-Fusobacterium Strains 59 3.3.2 Average Nucleotide Identity 59 3.3.3 MUMmer and MUMi 60 3.3.4 Development of Pan-Locus Sequence Analysis 61 3.3.5 Score Comparison 62 3.4 Defining the Taxonomic Boundary 64 3.5 Reclassification of Multiple Species 67 3.5.1 Identification and Classification of F.
Load more

Recommended publications
  • MALDI-TOF MS for the Identification of Anaerobic Bacteria
    University Medical Center Groningen Department Medical Microbiology and Infection Prevention Linda Veloo Expert Center Anaerobic Infections The Netherlands www.mmb-umcg.eu © by author ESCMID Online Lecture Library Matrix Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS) Time of flight tube Target at 15-25 kV Detector Ion source © by author ESCMID- “Time of flight” Online of individual Lecture proteins is converted Library into mass information. - Spectrum is produced - Database is built Veloo et al. Anaerobe 2011; 17:211-212 Workflow Direct spotting of bacteria on target using a toothpick Add HCCA matrix Data acquisition © Databy analyses author ESCMID Online Lecture Library Log score: <1.7 no reliable identification 1.7 – 2.0 reliable genus identification ≥ 2.0 reliable species identification Obtained spectrum is unique for bacterial species Intensity © by author ESCMID Online Lecture Library Anaerobic culture Phenotypic pure culture primary incubation 2 days 2-7 days aerotolerance identification MALDI-TOF MS 1 day 2-14 days primary incubation 2-7 days © by author MALDI-TOF MS testing minutes ESCMID Online Lecture Library How many anaerobic bacteria can be identified using MALDI-TOF MS? UMCG 2011/2012 Total no. of strains 1000 Species ID 650 65% Genus ID 149 15% No ID © 201by author20% ESCMID Online Lecture Library Performance differs per genus Genus* % species ID % genus ID Clostridium sp. (n=149) 97 3 B. fragilis sp. (n=179) 97 3 Parabacteroides sp. (n=14) 93 7 GPAC (n=133) 85 15 Prevotella sp.(n=83) 78 12 Propionibacterium sp. (n=129) 64 36 Actinomyces sp. (n=28) 57 43 Fusobacterium sp.
    [Show full text]
  • Microbiota of Human Precolostrum and Its Potential Role As a Source Of
    www.nature.com/scientificreports OPEN Microbiota of human precolostrum and its potential role as a source of bacteria to the infant mouth Received: 24 October 2018 Lorena Ruiz1,2, Rodrigo Bacigalupe3, Cristina García-Carral2,4, Alba Boix-Amoros3, Accepted: 2 April 2019 Héctor Argüello5, Camilla Beatriz Silva2,6, Maria de los Angeles Checa7, Alex Mira3 & Published: xx xx xxxx Juan M. Rodríguez 2 Human milk represents a source of bacteria for the initial establishment of the oral (and gut) microbiomes in the breastfed infant, however, the origin of bacteria in human milk remains largely unknown. While some evidence points towards a possible endogenous enteromammary route, other authors have suggested that bacteria in human milk are contaminants from the skin or the breastfed infant mouth. In this work 16S rRNA sequencing and bacterial culturing and isolation was performed to analyze the microbiota on maternal precolostrum samples, collected from pregnant women before delivery, and on oral samples collected from the corresponding infants. The structure of both ecosystems demonstrated a high proportion of taxa consistently shared among ecosystems, Streptococcus spp. and Staphylococcus spp. being the most abundant. Whole genome sequencing on those isolates that, belonging to the same species, were isolated from both the maternal and infant samples in the same mother-infant pair, evidenced that in 8 out of 10 pairs both isolates were >99.9% identical at nucleotide level. The presence of typical oral bacteria in precolostrum before contact with the newborn indicates that they are not a contamination from the infant, and suggests that at least some oral bacteria reach the infant’s mouth through breastfeeding.
    [Show full text]
  • Identification of Anaerobic Gram Negative Rods
    UK Standards for Microbiology Investigations 2014 Identification of Anaerobic Gram Negative Rods FEBRUARY 24 - JANUARY 24 BETWEEN ON CONSULTED WAS DOCUMENT THIS - DRAFT Issued by the Standards Unit, Microbiology Services, PHE Bacteriology – Identification | ID 25 | Issue no: di+ | Issue date: dd.mm.yy <tab+enter> | Page: 1 of 21 © Crown copyright 2013 Identification of Anaerobic Gram Negative Rods Acknowledgments UK Standards for Microbiology Investigations (SMIs) are developed under the auspices of Public Health England (PHE) working in partnership with the National Health Service (NHS), Public Health Wales and with the professional organisations whose logos are displayed below and listed on the website http://www.hpa.org.uk/SMI/Partnerships. SMIs are developed, reviewed and revised by various working groups which are overseen by a steering committee (see http://www.hpa.org.uk/SMI/WorkingGroups). The contributions of many individuals in clinical, specialist and reference laboratories2014 who have provided information and comments during the development of this document are acknowledged. We are grateful to the Medical Editors for editing the medical content. For further information please contact us at: FEBRUARY 24 Standards Unit - Microbiology Services Public Health England 61 Colindale Avenue London NW9 5EQ JANUARY E-mail: [email protected] 24 Website: http://www.hpa.org.uk/SMI UK Standards for Microbiology Investigations are produced in association with: BETWEEN ON CONSULTED WAS DOCUMENT THIS - DRAFT Bacteriology – Identification | ID 25 | Issue no: di+ | Issue date: dd.mm.yy <tab+enter> | Page: 2 of 21 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England Identification of Anaerobic Gram Negative Rods Contents ACKNOWLEDGMENTS .........................................................................................................
    [Show full text]
  • Growth Requirements and Fermentation Products of Fusobacterium Prausnitzii, and a Proposal to Reclassify It As Faecalibacterium Prausnitzii Gen
    International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2141–2146 DOI: 10.1099/ijs.0.02241-0 Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov. 1 Division of Gut Sylvia H. Duncan,1 Georgina L. Hold,1 Hermie J. M. Harmsen,2 Microbiology and 1 1 Immunology, Rowett Colin S. Stewart and Harry J. Flint Research Institute, Greenburn Road, Bucksburn, Aberdeen Author for correspondence: Sylvia H. Duncan. Tel: j44 1224 712751. Fax: j44 1224 716687. AB21 9SB, UK e-mail: shd!rri.sari.ac.uk 2 Department of Medical Microbiology, University of Groningen, Groningen, Two newly isolated strains of obligately anaerobic bacteria from human faeces The Netherlands are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon. These strains, along with Fusobacterium prausnitzii ATCC 27768T and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products. A new finding is that all four strains produce D-lactate, but not L- lactate. The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid. The DNA GMC content of the four strains is 47–57 mol%. Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group). It is proposed that a new genus, Faecalibacterium gen.
    [Show full text]
  • Clinical Characteristics of Fusobacterial Brain Abscess
    Jpn. J. Infect. Dis., 60, 40-44, 2007 Original Article Clinical Characteristics of Fusobacterial Brain Abscess Mei-Jen Hsieh, Wen-Neng Chang, Chun-Chung Lui1, Chi-Ren Huang, Yao-Chung Chuang, Shu-Fang Chen, Chuei-Shiun Li and Cheng-Hsien Lu* Department of Neurology and 1Department of Radiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (Received August 8, 2006. Accepted November 27, 2006) SUMMARY: We retrospectively reviewed 122 patients with culture-proven bacterial brain abscesses (BBA) at our hospital over a period of 20 years and identified seven fusobacterial brain abscess patients. Here we describe the therapeutic experience in fusobacterial BBA cases and compare the clinical features of patients with single pathogen infection between fusobacterial and non-fusobacterial brain abscesses. Fusobacterium spp. accounted for 6% of the implicated pathogens of monomicrobial BBA. All seven fusobacterial brain abscess patients contracted the infection spontaneously, and two cases had important preceding events. F. nucleatum was the commonest one of the species described. Clinical presentations and laboratory data of these seven patients were similar to those of non-fusobacterial BBA, and in these patients the diagnosis was only confirmed by positive culture results. All seven patients were successfully treated with combined surgical and antimicrobial therapy. Although the average age tends to be older and there is a higher prevalence of multiloculated brain abscesses in patients with this type of BBA, the therapeutic outcome can be favorable with early diagnosis and prompt treatment. Kaohsiung, the largest medical center in southern Taiwan, is INTRODUCTION a 2,482-bed teaching hospital that serves as a primary and A brain abscess is a focal brain parenchymal infection tertiary referral care teaching hospital.
    [Show full text]
  • Faecalibacterium Prausnitzii: from Microbiology to Diagnostics and Prognostics
    The ISME Journal (2017) 11, 841–852 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej MINI REVIEW Faecalibacterium prausnitzii: from microbiology to diagnostics and prognostics Mireia Lopez-Siles1, Sylvia H Duncan2, L Jesús Garcia-Gil1 and Margarita Martinez-Medina1 1Laboratori de Microbiologia Molecular, Departament de Biologia, Universitat de Girona, Girona, Spain and 2Microbiology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK There is an increasing interest in Faecalibacterium prausnitzii, one of the most abundant bacterial species found in the gut, given its potentially important role in promoting gut health. Although some studies have phenotypically characterized strains of this species, it remains a challenge to determine which factors have a key role in maintaining the abundance of this bacterium in the gut. Besides, phylogenetic analysis has shown that at least two different F. prausnitzii phylogroups can be found within this species and their distribution is different between healthy subjects and patients with gut disorders. It also remains unknown whether or not there are other phylogroups within this species, and also if other Faecalibacterium species exist. Finally, many studies have shown that F. prausnitzii abundance is reduced in different intestinal disorders. It has been proposed that F. prausnitzii monitoring may therefore serve as biomarker to assist in gut diseases diagnostics. In this mini- review, we aim to serve as an overview of F. prausnitzii phylogeny, ecophysiology and diversity. In addition, strategies to modulate the abundance of F. prausnitzii in the gut as well as its application as a biomarker for diagnostics and prognostics of gut diseases are discussed.
    [Show full text]
  • Laboratory Diagnosis of Sexually Transmitted Infections, Including Human Immunodeficiency Virus
    Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus human immunodeficiency including Laboratory transmitted infections, diagnosis of sexually Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus Editor-in-Chief Magnus Unemo Editors Ronald Ballard, Catherine Ison, David Lewis, Francis Ndowa, Rosanna Peeling For more information, please contact: Department of Reproductive Health and Research World Health Organization Avenue Appia 20, CH-1211 Geneva 27, Switzerland ISBN 978 92 4 150584 0 Fax: +41 22 791 4171 E-mail: [email protected] www.who.int/reproductivehealth 7892419 505840 WHO_STI-HIV_lab_manual_cover_final_spread_revised.indd 1 02/07/2013 14:45 Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus Editor-in-Chief Magnus Unemo Editors Ronald Ballard Catherine Ison David Lewis Francis Ndowa Rosanna Peeling WHO Library Cataloguing-in-Publication Data Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus / edited by Magnus Unemo … [et al]. 1.Sexually transmitted diseases – diagnosis. 2.HIV infections – diagnosis. 3.Diagnostic techniques and procedures. 4.Laboratories. I.Unemo, Magnus. II.Ballard, Ronald. III.Ison, Catherine. IV.Lewis, David. V.Ndowa, Francis. VI.Peeling, Rosanna. VII.World Health Organization. ISBN 978 92 4 150584 0 (NLM classification: WC 503.1) © World Health Organization 2013 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for non-commercial distribution – should be addressed to WHO Press through the WHO web site (www.who.int/about/licensing/copyright_form/en/index.html).
    [Show full text]
  • Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors
    Published OnlineFirst May 8, 2017; DOI: 10.1158/1940-6207.CAPR-16-0178 Research Article Cancer Prevention Research Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors Xiangcang Ye1, Rui Wang1, Rajat Bhattacharya1, Delphine R. Boulbes1, Fan Fan1, Ling Xia1, Harish Adoni1, Nadim J. Ajami2, Matthew C. Wong2, Daniel P. Smith2, Joseph F. Petrosino2, Susan Venable3, Wei Qiao4, Veera Baladandayuthapani4, Dipen Maru5, and Lee M. Ellis1,6 Abstract Chronic infection and associated inflammation have using immunoassays and found that expression of the long been suspected to promote human carcinogenesis. cytokines IL17A and TNFa was markedly increased but Recently, certain gut bacteria, including some in the Fuso- IL21 decreased in the colorectal tumors. Furthermore, the bacterium genus, have been implicated in playing a role in chemokine (C-C motif) ligand 20 was differentially human colorectal cancer development. However, the Fuso- expressed in colorectal tumors at all stages. In in vitro co- bacterium species and subspecies involved and their onco- culture assays, F. nucleatum ssp. animalis induced CCL20 genic mechanisms remain to be determined. We sought to protein expression in colorectal cancer cells and monocytes. identify the specific Fusobacterium spp. and ssp. in clinical It also stimulated the monocyte/macrophage activation and colorectal cancer specimens by targeted sequencing of migration. Our observations suggested that infection with Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium F. nucleatum ssp. animalis in colorectal tissue could induce spp. were identified in clinical colorectal cancer specimens. inflammatory response and promote colorectal cancer Additional analyses confirmed that Fusobacterium nuclea- development. Further studies are warranted to determine tum ssp.
    [Show full text]
  • University of Ghana College of Basic and Applied Sciences by Shirley Victoria Simpson (10551058) This Thesis Is Submitted To
    UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES ISOLATION AND CHARACTERIZATION of Haemophilus ducreyi STRAINS FROM CHILDREN WITH CUTANEOUS LESIONS IN YAWS ENDEMIC REGIONS, GHANA BY SHIRLEY VICTORIA SIMPSON (10551058) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE JULY, 2017 DECLARATION This is to certify that this thesis is the result of research undertaken by me, Shirley Victoria Simpson towards the award of Master of Philosophy in Molecular Cell Biology of Infectious Diseases in the Department of Biochemistry, Cell and Molecular Biology, School of Biological Sciences, College of Basic And Applied Sciences, University of Ghana. Signature--------------------------------------- Date-------------------------- Shirley Victoria Simpson (Candidate) Signature--------------------------------------- Date----------------------- Prof. Kennedy Kwasi Addo (Supervisor) Signature------------------------------------ Date----------------------- Dr. Lydia Mosi (Co-Supervisor) i ABSTRACT Recent discovery of cutaneous H. ducreyi has complicated the epidemiology of Yaws in endemic countries. Yaws and H. ducreyi ulcers are clinically indistinguishable from each other and some other causes of skin ulcerations. The aim of the study was to isolate and characterize H. ducreyi strains from lesions of children in yaws-endemic areas. Symptomatic patients were first screened with Dual Path Platform (DPP-RDT) Syphilis Screen & Confirm test kit (Chembio, Medford, New York) for yaws. Lesion exudates were tested by culture for H. ducreyi and real-time multiplex PCR assays were used to identify T.p subsp. pertenue DNA and H. ducreyi DNA. Azithromycin (AZT) resistance markers were screened for in T.p subsp. pertenue PCR positives. Bacterial 16S rRNA gene was amplified and sequenced to detect the presence of other pathogenic bacteria.
    [Show full text]
  • An Insight Into Pericoronitis REVIEW ARTICLE
    Dhonge RP et al: An Insight Into Pericoronitis REVIEW ARTICLE An Insight into Pericoronitis Roshan P. Dhonge1, R. M. Zade2, V. Gopinath3, Ramesh Amirisetty4 1- Post Graduate Student, Department of Periodontology, Chhattisgarh Dental College And Research Institute, Rajnandgaon, Chhattisgarh (India). 2- Professor, HOD and Dean, Department of Periodontology, Chhattisgarh Dental College And Research Correspondence to: Institute, Rajnandgaon, Chhattisgarh (India). 3- Professor, Department of Dr. Roshan P. Dhonge, 10, Ganesh Vihar No. I, Near Periodontology, Chhattisgarh Dental College And Research Institute, Rajnandgaon, Swastik Nagar and Water Tank, Amravati, Maharashtra, Chhattisgarh (India). 4- Reader, Department of Periodontology, Chhattisgarh Dental India. College And Research Institute, Rajnandgaon, Chhattisgarh (India). ABSTRACT Pericoronitis is inflammation of the soft tissue associated with the crown of a partially erupted tooth. It is seen most commonly in relation to the mandibular third molar. The common symptoms and signs are pain, swelling, trismus, halitosis, bad taste, inflammation of pericoronal flap and pus discharge from underneath it, inflammation sometimes aggravated by trauma from an antagonist tooth. In severe episodes, an acute pericoronal abscess may develop which may remain localized or spread to involve one or more of the adjacent deep surgical spaces and may be associated with systemic as well as local signs and symptoms. The treatment for acute phase include debridement of plaque and food debris, drainage of pus, irrigation with sterile saline, chlorhexidine or hydrogen peroxide, elimination of occlusal trauma and prophylactic antibiotic along with analgesics. The treatment planning for surgical intervention will be made after acute phase subsided. An extraction of partially or completely impacted third molar should be done.
    [Show full text]
  • Microbiota Network and Mathematic Microbe Mutualism in Colostrum and Mature Milk Collected in Two Different Geographic Areas: Italy Versus Burundi
    The ISME Journal (2017) 11, 875–884 OPEN © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE Microbiota network and mathematic microbe mutualism in colostrum and mature milk collected in two different geographic areas: Italy versus Burundi Lorenzo Drago1,2, Marco Toscano1, Roberta De Grandi2, Enzo Grossi3, Ezio M Padovani4 and Diego G Peroni5,6 1Clinical Chemistry and Microbiology Laboratory, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy; 2Clinical Microbiology Laboratory, Department of Biomedical Science for Health, University of Milan, Milan, Italy; 3Villa Santa Maria Institute, Via IV Novembre Tavernerio, Como, Italy; 4Pediatric Department, University of Verona & Pro-Africa Foundation, Verona, Italy; 5Department of Clinical and Experimental Medicine, Section of Pediatrics, University of Pisa, Pisa, Italy and 6International Inflammation (in-FLAME) Network of the World Universities Network, Sydney, NSW, Australia Human milk is essential for the initial development of newborns, as it provides all nutrients and vitamins, such as vitamin D, and represents a great source of commensal bacteria. Here we explore the microbiota network of colostrum and mature milk of Italian and Burundian mothers using the auto contractive map (AutoCM), a new methodology based on artificial neural network (ANN) architecture. We were able to demonstrate the microbiota of human milk to be a dynamic, and complex, ecosystem with different bacterial networks among different populations containing diverse microbial hubs and central nodes, which change during the transition from colostrum to mature milk. Furthermore, a greater abundance of anaerobic intestinal bacteria in mature milk compared with colostrum samples has been observed. The association of complex mathematic systems such as ANN and AutoCM adopted to metagenomics analysis represents an innovative approach to investigate in detail specific bacterial interactions in biological samples.
    [Show full text]
  • Fusobacterium Nucleatum, Strain CTI-03 Catalog No. HM-994 for Research Use Only. Not for Human Use
    Product Information Sheet for HM-994 Fusobacterium nucleatum, Strain CTI-03 immediately upon arrival. For long-term storage, the vapor phase of a liquid nitrogen freezer is recommended. Freeze- thaw cycles should be avoided. Catalog No. HM-994 Growth Conditions: For research use only. Not for human use. Media: Modified Chopped Meat medium or equivalent Contributor: Tryptic Soy agar with 5% defibrinated sheep blood or Wendy S. Garrett, M.D., Ph.D., Assistant Professor, and equivalent Aleksander D. Kostic, Department of Immunology and Incubation: Infectious Diseases, Harvard School of Public Health, Boston, Temperature: 37°C Massachusetts, USA Atmosphere: Anaerobic Propagation: Manufacturer: 1. Keep vial frozen until ready for use, then thaw. BEI Resources 2. Transfer the entire thawed aliquot into a single tube of broth. Product Description: 3. Use several drops of the suspension to inoculate an Bacteria Classification: Fusobacteriaceae, Fusobacterium agar slant and/or plate. Species: Fusobacterium nucleatum 4. Incubate the tube, slant and/or plate at 37°C for 24 to 72 Strain: CTI-03 hours. Original Source: Fusobacterium nucleatum (F. nucleatum), strain CTI-03 was isolated in 2012 from colonic tumor tissue Citation: from a human patient with colorectal carcinoma in Acknowledgment for publications should read “The following Massachusetts, USA.1 reagent was obtained through BEI Resources, NIAID, NIH as Comments: F. nucleatum, strain CTI-03 (HMP ID 1540) is a part of the Human Microbiome Project: Fusobacterium reference genome for The Human Microbiome Project nucleatum, Strain CTI-03, HM-994.” (HMP). HMP is an initiative to identify and characterize human microbial flora. The complete genome of F.
    [Show full text]