DOCSLIB.ORG

Development of Novel Tools for Prevention and Diagnosis of Porphyromonas Gingivalis Infection and Periodontitis I Dedicate This Thesis to My Beloved Parents

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Development of Novel Tools for Prevention and Diagnosis of Porphyromonas Gingivalis Infection and Periodontitis I Dedicate This Thesis to My Beloved Parents Development of Novel Tools for Prevention And Diagnosis of Porphyromonas gingivalis Infection and Periodontitis I dedicate this thesis to my beloved parents ¨A small body of determined spirits fired by an unquenchable faith in their mission can alter the course of history¨ - Gandhi Örebro Studies in Medicine 151 SRAVYA SOWDAMINI NAKKA Development of Novel Tools for Prevention and Diagnosis of Porphyromonas gingivalis Infection and Periodontitis © Sravya Sowdamini Nakka, 2016 Title: Development of Novel Tools for Prevention and Diagnosis of Porphyromonas gingivalis Infection and Periodontitis Publisher: Örebro University 2016 www.publications.oru.se Print: Örebro University, Repro 09/2016 ISSN 1652-4063 ISBN 978-91-7668-162-8 Abstract Sravya Sowdamini Nakka (2016): Development of Novel Tools for Prevention and Diagnosis of Porphyromonas gingivalis Infection and Periodontitis. Örebro studies in Medicine 151. Periodontitis is a chronic inflammatory disease caused by exaggerated host im- mune responses to dysregulated microbiota in dental biofilms leading to degrada- tion of tissues and alveolar bone loss. Porphyromonas gingivalis is a major perio- dontal pathogen and expresses several potent virulence factors. Among these fac- tors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingi- valis infection and periodontitis. In study I, anti-P. gingivalis antibodies were de- veloped in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the re- sults were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lac- tobacillus species and the two-peptide bacteriocin PLNC8 on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 induced bacterial lysis by damaging P. gingivalisαβ membrane. In study III, we demonstrated that PLNC8 dose-dependently induces proliferation and release ofαβ growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 decreased P. gingivalis-induced αβcytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects ofαβ anti-P. gingivalis antibodies and PLNC8 in regulating cellular responses dur- ing P. gingivalis infection. Both antibodies and PLNC8 modulated P. gingi- valis-induced expression of growth factorsαβ in GECs, however, their effects were diminished when used in combination. The results of thisαβ thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells. αβ Keywords: Periodontitis, Porphyromonas gingivalis, anti-P. gingivalis antibodies, surface plasmon resonance, PLNC8 , proliferation, growth factors. Sravya Sowdamini Nakka, Örebro αβstudies in Medicine, Örebro University, SE-70182 Örebro, Sweden, Email: [email protected] Table of Contents LIST OF PAPERS ...................................................................................... 9 PUBLICATIONS NOT INCLUDED IN THIS STUDY ........................... 10 LIST OF ABBREVIATIONS ................................................................... 11 INTRODUCTION .................................................................................. 13 Periodontitis ............................................................................................ 13 Porphyromonas gingivalis ....................................................................... 14 Oral Biofilms ........................................................................................... 16 Periodontitis and P. gingivalis Associated Systemic Diseases ................... 16 Host Immune Responses ......................................................................... 17 Antibodies ............................................................................................... 20 Diagnosis, Treatment and Prevention of Periodontitis ............................ 21 Lactobacillus and Bacteriocins ................................................................ 22 Aims ........................................................................................................ 25 METHODS ............................................................................................. 26 Study Participants and Ethical Approval ................................................. 26 Bacterial Strains and Bacteriocins ............................................................ 26 Cell Culturing ......................................................................................... 27 Antibody Production ............................................................................... 27 Flow Cytometry ...................................................................................... 28 Transmission Electron Microscopy ......................................................... 28 Confocal Microscopy .............................................................................. 29 Surface Plasmon Resonance Analysis ...................................................... 29 DNA-DNA Hybridization Technique ..................................................... 30 Enzyme-linked Immunosorbent Assay ..................................................... 31 Growth Factor Array .............................................................................. 31 Liposome Model ..................................................................................... 32 Antibacterial Activity of PLNC8 ........................................................ 32 Circular Dichroism Spectroscopy ............................................................ 33 Western Blotting ................................αβ ..................................................... 33 Statistics .................................................................................................. 34 RESULTS AND DISCUSSION ................................................................ 35 Activation of Leukocytes and Induction of Secondary Immune Responses (Study I) .................................................................................................. 35 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 7 Detection and Attenuation of P. gingivalis by Anti-P. gingivalis antibodies (Study I) ................................................................................................... 36 Antibacterial Activity of Lactobacillus spp. and PLNC8 αβ (Study II) .... 36 Effects of PLNC8 αβ per se on GECs and During P. gingivalis Infection (Studies III & IV) ..................................................................................... 37 Effects of Anti-P. gingivalis Antibodies on GECs and During P. gingivalis Infection (Study IV) ................................................................................. 39 Combined Effects of Anti-P. gingivalis Antibodies and PLNC8 αβ on P. gingivalis-infected GECs (Study IV) .................................................... 40 CONCLUSIONS ..................................................................................... 41 FUTURE PERSPECTIVES ....................................................................... 42 ACKNOWLEDGEMENTS ..................................................................... 43 REFERENCES ........................................................................................ 46 8 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection List of Papers This thesis is based on the following original papers and manuscripts, which are referred to in the text by their roman numerals: I. Sravya Sowdamini Nakka, Johanna Lönn, Carin Starkhammar Jo- hansson, Torbjörn Bengtsson and Fariba Nayeri. Antibodies pro- duced in vitro in the detection of periodontal bacteria by using sur- face plasmon resonance analysis. Clinical and experimental dental research 2015, 1:32. II. Hazem Khalaf, Sravya Sowdamini Nakka, Camilla Sandén, Anna Svärd, Kjell Hultenby, Nikolai Scherbak, Daniel Aili and Torbjörn Bengtsson. Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingi- valis. BMC Microbiology 2016, 16:188. III. Sravya Sowdamini Nakka, Eleonor Palm, Torbjörn Bengtsson, Hazem Khalaf. Bacteriocin plantaricin NC8 αβ antagonizes Por- phyromonas gingivalis infection and induces proliferation of gin- gival epithelial cells. (Manuscript). IV. Sravya Sowdamini Nakka, Eleonor Palm, Fariba Nayeri, Torbjörn Bengtsson, Hazem Khalaf. Effects of plantaricin NC8 αβ and an- tibodies on gingival epithelial cells infected by Porphyromonas gin- givalis. (Manuscript). SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 9 Publications not included in this study Chongcong Wu, Sravya Sowdamini Nakka, Sepahdar Mansouri, Tor- björn Bengtsson, Tayeb Nayeri and Fariba Nayeri. In vitro model of pro- duction of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments. BMC Microbiology 2016 (in press). Johanna Lönn, Carin Starkhammar Johansson, Sravya Sowdamini Nakka, Eleonor Palm, Torbjörn Bengtsson, Fariba Nayeri F, Nils Ravald.
Load more

Recommended publications
  • Dynamic Dashboard - List of Bacteria with a Relevant AMR Issue
    14 May 2020 Dynamic Dashboard - List of bacteria with a relevant AMR issue Categorization and inclusion methodology for human bacterial pathogens The World Health Organization’s (WHO) Global Priority List of Antibiotic-Resistant Bacteria [1], the United States of America’s Centers for Disease Control and Prevention (CDC) Antibiotic Resistant Threats in the United States 2019 [2] and the bacteria included in the European Centre for Disease Prevention and Control’s (ECDC) European Antimicrobial Resistance Surveillance Network (EARS-Net) [3] were used to develop a combined list of priority bacteria with a drug-resistance issue. It was considered that all research into these bacteria, irrespective of the drug-resistance profile, would be relevant to advance efforts to address antimicrobial resistance. For the categorization process, only the bacterial genus level (noting the family Enterobacteriaceae was also included) was used and will be displayed. Table 1 list the genus included in categorization and the rules applied for inclusion based on the aforementioned priority lists. When projects included bacteria other than those listed in Table 1 an individual assessment was performed by the Secretariat to determine if there is a known drug-resistance issue. This assessment included searching the literature and reaching a consensus within the Secretariat if the bacteria has a known drug- resistance issue or not. Where consensus was not reached or there was ambiguity in the literature bacteria were parked and further investigation was conducted. The list of additional bacteria and the outcomes from the assessment are provided in Table 2 and 3. Please note that these lists will be continually updated.
    [Show full text]
  • Date Last Revised
    CURRICULUM VITAE NAME: Catherine L. Carlson Haggerty, Ph.D., M.P.H. BUSINESS Department of Epidemiology ADDRESS: 5133 Public Health Graduate School of Public Health University of Pittsburgh 130 DeSoto Street Pittsburgh, PA 15261 Phone: (412) 624-7377 Fax: (412) 624-7397 Email: [email protected] Webpage: http://www.publichealth.pitt.edu/home/directory/catherine-l- haggerty EDUCATION AND TRAINING Undergraduate 1990-1994 University of Pittsburgh, B.S. Mathematics Johnstown, PA Graduate 1996-1999 University of Pittsburgh M.P.H Epidemiology Graduate School of Public Health Pittsburgh, PA 1996-2001 University of Pittsburgh Ph.D. Epidemiology Graduate School of Public Health Pittsburgh, PA Post-Graduate 2002 University of Pittsburgh Community Health Summer Internship Mentor: Ken Jaros, Graduate School of Placement: COTRAIC Early Head Start Ph.D., M.S.W Public Health and Pittsburgh Family Center for Child Pittsburgh, PA Development Page 1 of 82 2001-2004 University of Pittsburgh NIH NRSA Mentor: Roberta Ness, Graduate School of Individual Training Fellowship M.D., M.P.H Public Health Department of Epidemiology Pittsburgh, PA APPOINTMENTS AND POSITIONS Academic 2011-present Associate Professor of Department of Epidemiology Reproductive Epidemiology Graduate School of Public Health with Tenure University of Pittsburgh, Pittsburgh, PA 2008-present Director Department of Epidemiology Reproductive, Perinatal & Graduate School of Public Health Pediatric Epidemiology Area of University of Pittsburgh, Pittsburgh, PA Emphasis 2003-present Affiliate Faculty
    [Show full text]
  • Genomic Islands in Mycoplasmas
    G C A T T A C G G C A T genes Review Genomic Islands in Mycoplasmas Christine Citti * , Eric Baranowski * , Emilie Dordet-Frisoni, Marion Faucher and Laurent-Xavier Nouvel Interactions Hôtes-Agents Pathogènes (IHAP), Université de Toulouse, INRAE, ENVT, 31300 Toulouse, France; [email protected] (E.D.-F.); [email protected] (M.F.); [email protected] (L.-X.N.) * Correspondence: [email protected] (C.C.); [email protected] (E.B.) Received: 30 June 2020; Accepted: 20 July 2020; Published: 22 July 2020 Abstract: Bacteria of the Mycoplasma genus are characterized by the lack of a cell-wall, the use of UGA as tryptophan codon instead of a universal stop, and their simplified metabolic pathways. Most of these features are due to the small-size and limited-content of their genomes (580–1840 Kbp; 482–2050 CDS). Yet, the Mycoplasma genus encompasses over 200 species living in close contact with a wide range of animal hosts and man. These include pathogens, pathobionts, or commensals that have retained the full capacity to synthesize DNA, RNA, and all proteins required to sustain a parasitic life-style, with most being able to grow under laboratory conditions without host cells. Over the last 10 years, comparative genome analyses of multiple species and strains unveiled some of the dynamics of mycoplasma genomes. This review summarizes our current knowledge of genomic islands (GIs) found in mycoplasmas, with a focus on pathogenicity islands, integrative and conjugative elements (ICEs), and prophages. Here, we discuss how GIs contribute to the dynamics of mycoplasma genomes and how they participate in the evolution of these minimal organisms.
    [Show full text]
  • Mycoplasma Genitalium, a New Species from the Human Urogenital Tract
    INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1983, p. 387-396 Vol. 33. No. 2 0020-7713/83/020387-10$02.0010 Mycoplasma genitalium, a New Species from the Human Urogenital Tract JOSEPH G. TULLY,'* DAVID TAYLOR-ROBINSON,3DAVID L. ROSE,' ROGER M. COLE,' AND JOSEPH M. BOVE4 Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, FCRS, Frederick, Maryland 21 701'; Laboratory of Streptococcal Diseases,' National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 202052; Medical Research Council Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex, England3; and Laboratoire de Biologie Cellulaire et Moleculaire, Institut National de la Recherche Agronomique, and University of Bordeaux 11, Domaine de la Grande Ferrade, 33 140 Pont-de-la-May e, France4 Two mycoplasmas recovered from human urogenital tracts were similar in their biochemical and serological properties. These organisms possessed a unique terminal structure that appeared to be associated with attachment to tissue cells and erythrocytes. The organisms fermented glucose but did not hydrolyze urea or arginine. Growth occurred at 30 to 37°C. Cholesterol was required for growth. Unlike most other mycoplasmas, both strains were susceptible to thallium acetate. These two organisms were serologically distinct from other Mycoplasma species and from a group of unclassified serotypes of mycoplasmas. On the basis of these findings and other morphological, biological, and serological properties of the microorganisms, we propose that mycoplasmas with these characteristics belong to a new species, Mycoplasma genitalium. Strain G-37 (= ATCC 33530) is the type strain. It is thought that the mycoplasma flora of These and other observations suggest that humans consists of 11 distinct microorganisms, other more fastidious mycoplasmas or other including nine Mycoplasma species, Achole- tetracycline-susceptible microorganisms or both plasma laidlawii, and Ureaplasma urealyticrrm might be involved in NGU and perhaps other (6).
    [Show full text]
  • CDC: Deaths from Antibiotic-Resistant Infections Higher Than Previously
    CDC: Deaths from antibiotic-resistant infections higher than previously estimated by Melissa Jenco, News Content Editor The death toll from antibiotic-resistant infections is higher than previously estimated but is improving, federal health officials said Wednesday. The Centers for Disease Control and Prevention (CDC) found there are about 35,000 such deaths each year in the U.S. and 2.8 million infections, according to its new report, Antibiotic Resistance Threats in the United States, 2019. Officials urged continued vigilance. "Bacteria and fungi will continue to develop resistance to drugs designed to kill them and without continued action could undo the progress we are sharing this afternoon,"CDC Director Robert R. Redfield, M.D., said. The CDC's 2013 report estimated there were 23,000 deaths annually, but revised estimates using additional records indicate it was nearly twice that. Using the revised data, it found deaths from antibiotic-resistant infections have dropped 18%, largely due to a 28% reduction at hospitals. The report categorizes 18 antibiotic-resistant germs as urgent, serious or concerning. Two germs - drug- resistant Candida auris and carbapenem-resistant Acinetobacter -were added to the list of urgent threats that already included carbapenem-resistant Enterobacteriaceae (CRE), drug-resistant Neisseria gonorrhoeae and Clostridioides difficile (C. difficile). The CDC highlighted significant increases from 2013 to 2019, including drug-resistant Neisseria gonorrhoeae infections, which more than doubled to 55,000 annually, and erythromycin-resistant group A Streptococcus infections, which spiked from 1,300 to 5,400. Extended-spectrum beta-lactamase-producing Enterobacteriaceae also is on the rise, causing 197,400 infections and 9,100 deaths a year.
    [Show full text]
  • As M. Genitalium, Gonorrhea Molecular Testing Expands, Antibiotic Resistance Assays May Follow | 360Dx
    9/6/2019 As M. Genitalium, Gonorrhea Molecular Testing Expands, Antibiotic Resistance Assays May Follow | 360Dx Article: In-Depth As M. Genitalium, Gonorrhea Molecular Testing Expands, Antibiotic Resistance Assays May Follow has been updated. As M. Genitalium, Gonorrhea Molecular Testing Expands, Antibiotic Resistance Assays May Follow Sep 06, 2019 | Madeleine Johnson 3D illustration of M. genitalium NEW YORK – Discovered only in the 1980s, Mycoplasma genitalium is more common than gonorrhea and has higher levels of antibacterial resistance in some populations. But because it's relatively new sexually transmitted infection and can take up to six months to grow in culture, it is a less well-studied disease and its prevalence and pathology remain somewhat of a mystery. Newly available US Food and Drug Administration-cleared molecular diagnostic tests, as well as in- development commercial tests to detect resistance, however, may soon enable a better understanding of the epidemiology of drug-resistant STIs like M. gen and gonorrhea. But the market for the tests is unclear as such infections are typically more prevalent in disadvantaged populations — potentially making them less commercially attractive — and testing guidelines in the US have not been updated since 2015. Antibacterial resistance is a growing problem for sexually transmitted infections, and M. gen and gonorrhea are no exceptions. Approximately half of M. gen infections are resistant to antibiotics in some parts of the world, including in the US, and increasing rates of drug-resistant gonorrhea is also raising alarms. The US Food and Drug Administration-cleared molecular diagnostic tests to detect M. gen finally became available this year, from Roche and Hologic, and a test that detects genetic resistance markers in M.
    [Show full text]
  • Antibiotic Resistance Threats in the United States, 2019
    ANTIBIOTIC RESISTANCE THREATS IN THE UNITED STATES 2019 Revised Dec. 2019 This report is dedicated to the 48,700 families who lose a loved one each year to antibiotic resistance or Clostridioides difficile, and the countless healthcare providers, public health experts, innovators, and others who are fighting back with everything they have. Antibiotic Resistance Threats in the United States, 2019 (2019 AR Threats Report) is a publication of the Antibiotic Resistance Coordination and Strategy Unit within the Division of Healthcare Quality Promotion, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention. Suggested citation: CDC. Antibiotic Resistance Threats in the United States, 2019. Atlanta, GA: U.S. Department of Health and Human Services, CDC; 2019. Available online: The full 2019 AR Threats Report, including methods and appendices, is available online at www.cdc.gov/DrugResistance/Biggest-Threats.html. DOI: http://dx.doi.org/10.15620/cdc:82532. ii U.S. Centers for Disease Control and Prevention Contents FOREWORD .............................................................................................................................................V EXECUTIVE SUMMARY ........................................................................................................................ VII SECTION 1: THE THREAT OF ANTIBIOTIC RESISTANCE ....................................................................1 Introduction .................................................................................................................................................................3
    [Show full text]
  • The Epidemiology of Chlamydia Trachomatis and Mycoplasma Genitalium in Young Australian Women
    The epidemiology of Chlamydia trachomatis and Mycoplasma genitalium in young Australian women. Jennifer Walker Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy. January 2011 School of Population Health The University of Melbourne. ABSTRACT Chlamydia trachomatis is the most common notifiable sexually transmissible infection in young Australian women and has considerable public health consequences, yet the true burden of disease caused by chlamydia remains unknown. There are also no population data for Mycoplasma genitalium, another emerging sexually transmissible infection in young women. This thesis aims to address two issues relevant to C. trachomatis and M. genitalium . The first aim was to determine the effectiveness of an intervention in general practice to increase chlamydia testing rates, and the second aim was determine the community burden of both C. trachomatis and M. genitalium in young women in Australia. Chapter 4 describes the C-Alert study which was a randomised controlled trial to determine the effectiveness of a computer alert in general practice on C. trachomatis testing rates in young women. The computer alert increased testing by 27% [AOR:1.27 (95%CI: 1.11-1.45)], and this small but significant increase suggests alerts might be beneficial along with other interventions to increase C. trachomatis testing in general practice. Chapters 5, 6, 7, 8, 9 and 10 describe the CIRIS study which was a cohort study involving 1116 young Australian women. C. trachomatis prevalence at baseline was 4.9% (95%CI: 2.9-7.0) and incidence rate for the 12–month study period was 4.4 per 100 women-years (95%CI: 3.3-5.9).
    [Show full text]
  • Healthtrackrx Syndromic Menu, Symptoms and Order Chart
    HealthTrackRx Syndromic Menu, Symptoms and Order Chart HealthTrackRx is an industry leading infectious disease laboratory providing accurate, targeted molecular diagnostics. With over 4,000,000 samples processed and a turn around time within 24 hours of sample receipt, our technological advances in PCR testing reshape patient care by mobilizing faster clinical decisions and helping providers treat patients quickly and accurately. OPHTHALMOLOGY INFECTION OTOLARYNGOLOGY INFECTION SAMPLE TYPE: Eye Swab, Lacrimal Fluid, Vitreous Fluid SAMPLE TYPE: Ear Swab, Tympanocentesis Fluid, Nasopharynx Swab, Throat Swab Acinetobacter baumanii Moraxella catarrhalis Acinetobacter baumanii Klebsiella pneumoniae, oxytoca Adenovirus HAdV-B Mycoplasma genitalium, Adenovirus HAdV-B Moraxella catarrhalis Aspergillus flavus, fumigatus, hominis Aspergillus flavus, fumigatus, Mycoplasma genitalium, hominis niger, terreus Mycoplasma pneumoniae niger, terreus Mycoplasma pneumoniae Candida albicans, glabrata, Neisseria gonorrhoeae Candida albicans, glabrata, Neisseria gonorrhoeae parapsilosis, tropicalis Parainfluenza virus (types 1, 2, 3, 4) parapsilosis, tropicalis Parainfluenza virus (types 1, 2, 3, 4) Candida auris Proteus mirabilis, vulgaris Candida auris Proteus mirabilis, vulgaris Chlamydia pneumoniae Pseudomonas aeruginosa Chlamydia pneumoniae Pseudomonas aeruginosa Chlamydia trachomatis Respiratory syncytial virus Chlamydia trachomatis Respiratory syncytial virus Coronavirus (229E, NL63, OC43, Rhinovirus A, C Coronavirus (229E, NL63, OC43, Rhinovirus A,C
    [Show full text]
  • Number K190433 B Applicant Roch
    Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY I Background Information: A 510(k) Number K190433 B Applicant Roche Molecular Systems, Inc. C Proprietary and Established Names cobas TV/MG for use on cobas 6800/8800 systems, cobas TV/MG Positive Control Kit, cobas Buffer Negative Control Kit D Regulatory Information Product Regulation Classification Panel Code(s) Section 21 CFR 866.3393 - Device to Detect Nucleic Acids from Non-Viral QEP Class II Microorganism(s) Causing Sexually 83; MI - Microbiology Transmitted Infections and Associated Resistance Marker(s) II Submission/Device Overview: A Purpose for Submission: To determine substantial equivalence for the cobas TV/MG assay for use on cobas 6800/8800 systems for detection of Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) DNA in male or female urine, self-collected vaginal swab specimens, clinician-collected vaginal swab specimens, and endocervical specimens, for the detection of TV DNA in cervical specimens collected in PreservCyt solution and for the detection of MG DNA in self-collected meatal swab specimens and clinician-collected meatal swab specimens. B Measurand: Trichomonas vaginalis and Mycoplasma genitalium DNA K190433 - Page 1 of 41 C Type of Test: Nucleic acid extraction, purification and amplification assay (real-time polymerase chain reaction) III Intended Use/Indications for Use: A Intended Use(s): cobas TV/MG on the cobas 6800/8800 Systems is an automated, qualitative in vitro nucleic acid diagnostic test that utilizes real-time polymerase chain reaction (PCR), for the direct detection of Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) DNA in male or female urine, self-collected vaginal swab specimens (collected in a clinical setting), clinician-collected vaginal swab specimens, and endocervical specimens, all collected in cobas PCR Media (Roche Molecular Systems, Inc.).
    [Show full text]
  • Designing Minimal Genomes Using Whole-Cell Models
    ARTICLE https://doi.org/10.1038/s41467-020-14545-0 OPEN Designing minimal genomes using whole-cell models ✉ Joshua Rees-Garbutt1,2,7, Oliver Chalkley1,3,4,7, Sophie Landon1,3, Oliver Purcell5, Lucia Marucci1,3,6,8 & ✉ Claire Grierson1,2,8 In the future, entire genomes tailored to specific functions and environments could be designed using computational tools. However, computational tools for genome design are 1234567890():,; currently scarce. Here we present algorithms that enable the use of design-simulate-test cycles for genome design, using genome minimisation as a proof-of-concept. Minimal gen- omes are ideal for this purpose as they have a simple functional assay whether the cell replicates or not. We used the first (and currently only published) whole-cell model for the bacterium Mycoplasma genitalium. Our computational design-simulate-test cycles discovered novel in silico minimal genomes which, if biologically correct, predict in vivo genomes smaller than JCVI-Syn3.0; a bacterium with, currently, the smallest genome that can be grown in pure culture. In the process, we identified 10 low essential genes and produced evidence for at least two Mycoplasma genitalium in silico minimal genomes. This work brings combined computational and laboratory genome engineering a step closer. 1 BrisSynBio, University of Bristol, Bristol BS8 1TQ, UK. 2 School of Biological Sciences, University of Bristol, Bristol Life Sciences Building, 24 Tyndall Avenue, Bristol BS8 1TQ, UK. 3 Department of Engineering Mathematics, University of Bristol, Bristol BS8 1UB, UK. 4 Bristol Centre for Complexity Science, Department of Engineering Mathematics, University of Bristol, Bristol BS8 1UB, UK.
    [Show full text]
  • Identification of Essential Genes of the Periodontal Pathogen
    Klein et al. BMC Genomics 2012, 13:578 http://www.biomedcentral.com/1471-2164/13/578 RESEARCH ARTICLE Open Access Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis Brian A Klein1,2,ElizabethLTenorio2,DavidWLazinski1,3,AndrewCamilli1,3, Margaret J Duncan4 and Linden T Hu1,2* Abstract Background: Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. Results: In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P.
    [Show full text]