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Managing Outbreaks in an Era of Next Generation Sequencing International Meeting on Microbial Epidemiological Markers IMMEM-10 OCTOBER 2 – 5, 2013 Paris http://www.immem-10.org/ Abstract submission deadline: June 15th, 2013 Sessions: • Bioinformatics tools for genome-based microbial surveillance • Outbreak genomics and epidemiology • Population genetics, phylogenomics, emergence • Molecular typing and© epidemiology by author • Surveillance networks in practice • Phylodynamics of viral pathogens • Virulence: diagnostic and epidemiology ESCMID• Resistance: diagnosticOnline and epidemiology Lecture Library • Diagnostic by high-throughput sequencing • Social networks and transmission modelling • Strain tracking from global health to One Health • Round tables, workshops on various topics & networks Managing Outbreaks in an Era of Next Generation Sequencing © by author ESCMID Online Lecture Library Dag Harmsen University of Münster, Germany Short CV, Dag Harmsen • 1983–1991, Medical Schools University Antwerpen, Belgium and Univ. Würzburg, Germany • 1991, MD and doctoral thesis (“Nucleocapsid gene of Coronavirus HCV-229E”, V. ter Meulen) • 1992 – 2000, Institute of Hygiene & Microbiology (J. Heesemannn, Candida spp. & Aspergillus spp. & M. Frosch, Mycobacterium spp.) / Internal Medicine (K. Kochsiek) / Institute of Virology (V. ter Meulen), Univ. Würzburg, Germany • 1998, Inclusion in the German Microbiology and Infectious Epidemiology specialist register • 2000-2001, Head of R&D CREATOGEN diagnostics, CREATOGEN AG, Augsburg, Germany • 2002-2004, Institute of Hygiene (H. Karch, MRSA), University of Münster, Germany • 2003, co-founder and shareholder of a bioinformatics company (Ridom GmbH, Münster, Germany) • 2004– , Full Professorship Department of Periodontology, Univ. Münster, Germany • July 2005 – July 2008, Temporary Head of the Department of Periodontology, Univ. Münster • August 2008 – , Head of Research Department of Periodontology • July 2005 – , Member of the Executive© Board by of the author International Committee on Systematics of Prokaryotes (ICSP) of the "International Union of Microbiological Societies" (IUMS) • June 2007–2012, Member of the ASM Professional Development Committee • October 2012 - , ASM Ambassador in Germany ESCMID Online Lecture Library • Scientific interests: molecular diagnostic, epidemiology, and phylogeny of microorganisms; applied bioinformatics in microbiology Setting © by author ESCMID Online Lecture Library London, UK - 1854 © by author ESCMID Online Lecture Library John Snow – Broad Street Cholera Outbreak © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library ONE Health Needed ONE Health - "the collaborative effort of multiple disciplines — working locally, nationally, and globally — to attain optimal health for people, animals and the environment". The American Veterinary Medical© Association. by authorOne Health Initiative Task Force. "One Health: A New Professional Imperative". July 15, 2008. https://www.avma.org/KB/Resources/Reports/Documents/onehealth_final.pdf ESCMID Online Lecture Library Fourth Dimension Needed © by author ESCMID Online Lecture Library Place, Time, Person … Type! ONE Disruptive Technology Next Generation Sequencing - Bench-top Machines Roche/454 GS Junior Ion Torrent Personal Genome Machine (PGM©) by author ESCMID• Affordable Online Lecture Library • Speed illumina MiSeq Personal Sequencing System • Simple workflow Diagnostic Workflow in Clinical Microbiology Detection Identification Susceptibility Epidemiology * © by author ESCMID OnlineDidelot et al. (2012).Lecture Nature Rev. Genet. doi :Library 10.1038/nrg3226 [PubMed] number of samples *Mellmann et al. (2008). J. Clin. Microbiol. 46: 1946 [PubMed] Bench-top WGS NGS Achievements Prospective Genomic Epidemiology German EHEC Outbreak 2011 University Münster / Life Technologies ‘Tweetom‘ year 2011 week 21 22 23 24 25 26 27 28 29 30 © by author ESCMID Online Lecture Library BGI / UKE / Crowd-sourcing & HPA / Univ. Göttingen / Illumina / PacBio Phylogenetic Analysis of EHEC O104:H4 Phylogenetic analyses (by ‘quick and dirty’ hybrid RefMap & de novo assemblies & BIGSdb MLST+; n = 1.144 core genome genes, and minimum-spanning tree) • strain LB226692 (outbreak 2011) and strain 01-09591 (2001 German historic HUS causing isolate) belong to the HUSEC041 complex © by author• both strains are only distantly related to commonly isolated EHEC serotypes ESCMID Online Lecture Library Mellmann et al. (2011). PLoS One. 6: e22751 [PubMed] Timeline – Microbial Surveillance and Disease © by author Red boxes indicate disease outbreaks, blue boxes indicate technological advances, and green boxes indicate events relating to surveillance. E. coli, Escherichia coli; GPHIN, Global Public Health Intelligence Network;ESCMID InSTEDD, Innovative SupportOnline to Emergencies, Lecture Diseases and Disasters; Library ProMED-mail, Program for Monitoring Emerging Infectious Diseases; SARS, severe acute respiratory syndrome. Lipkin (2013). Nature Rev. Microbiol. 11: 133 [PubMed]. Rapid NGS Diagnostic Workflow in Clinical Microbiology • ‘ Ad hoc’ prospective genomic epidemiology technical feasible – by introduction of ‘bench-top’ Next Generation Sequencing (NGS) machines, prospective genomic epidemiology is achievable for small- and medium- sized laboratories (‘democratizing of NGS’) • Genomics for diagnostics – molecular diagnostic screening tests & ultra-deep sequencing for pathogen discovery from human tissues (e.g., hemorrhagic viruses) • Genomics for therapeutics – e.g., antibiotics, pathogenicity© by profiling, author and/or [reverse] vaccinology • Genomics for global surveillance and early-warning outbreakESCMID detection Online Lecture Library – Whole Genome Shotgun [WGS] NGS information-rich ‘typing’ Lesson to Be Learned From Previous Most Successful ‘Typing’ Networks © by author ESCMID Online Lecture Library Pulse Field Gel Electrophoresis (PFGE) Suspension of bacteria + Agarose Agar plugs Lyses (24h) Washing (24h) Equilibration (24 h) Restriction (24h) Pulsed field gel A B electrophoresis (24-48h) © by author B A Ethidium – + UV light ESCMIDbromide Online Lecture Library http://www.cdc.gov/pulsenet/ Multi Locus Sequence Typing (MLST) • Long term epidemiology, replaced MLEE completely • 5-7 housekeeping genes (selectively neutral) distri- buted over the whole genome (recombination independent http://pubmlst.org loci) • Sequence determination of 5-7 internal fragments (one Neisseria meningitidis sequencing run) of these genes • Identical gene sequences are assigned the sa me allele number • For each strain, the http:///www.mlst.net combination of alleles at each locus defines its sequence © by author type (ST) • Related S Ts are grouped Maiden et al. (1998). PNAS 95: 3140 [PubMed]. into clonal complexes (CC) • Single expanding central ESCMID Online LectureMLST Library databases that can be interrogated electronically, but Central public global nomenclature that is curated manually server (‘Molecular Typing Esperanto’). Spa typing (CE Sequencing SLST): StaphType & PubMed MLST.net Nomenclature & epidemiology server that is automatically quality based curated! SpaServer Ridom StaphType Client Synchronization (bidirectional communication) © by1. Newauthor global spa types & repeats 2. Submission of new local spa types & repeats 3. Submission of local frequencies & strain information 4. Delivery of global frequencies 5. Software updates ESCMID Online6. LectureMessages Library Submission of new The data to be synchronized is definable by every user! spa types via WWW Person data are never synchronized!!! (open system) http://www.SpaServer.ridom.de/ Harmsen et al. (2003). JCM 41: 5442 [PubMed]. Laboratory & Bioinformatics Challenges © by author ESCMID Online Lecture Library NGS Bottlenecks © by author ESCMID Online Lecture Library Sample processing NGS platform(s) tools, IT-infrastructure, & (bio)informaticians Laboratory Challenges © by author ESCMID Online Lecture Library Chain et al. (2009). Science. 326: 326 [PubMed] Draft genomes can be used to rule-in but NOT to definitively rule-out the existence of e.g. resistance and/or virulence genes Laboratory Developments Towards Finished Genomes • Fast & easy mate-pair protocols (insert > 5kb) • Hybrid 2nd- & 3rd generation assemblies Koren et al. (2012). Nature Biotechnology. doi: 10.1038/nbt.2280 [PubMed] © by author • HGAP – Hierarchical Genome ESCMIDAssembly Process Online (10kb Lecture seeds) Library Bioinformatics Challenge - ‘Molecular Typing Esperanto’ by Standardized Genome Comparison • Average Nucleotide Identity (ANI) / k-mer approach – used in taxonomy for species delineation, no assembly needed (suitable for clone level?) – whole genome finally reduced to a single number of (dis)similarity • Pair -wise (visual) genome comparison (Mauve-like) – in general very problematic due to genomic rearrangement and recombination events; (nearly) impossible with draft genomes and more than 40 genomes • Genome-wide SNP approach – works especially good for ‘ad hoc’ analysis of monomorphic organisms; various very ‘subjective’ SNP© byselection author approaches that are difficult to reproduce • Genome-wide gene by gene approach –ESCMIDworks on gene level, Online i.e. the element Lecture of evolution Library – sort of ‘super/extended’ MLST (MLST+), hierarchical typing approaches possible – enables potentially for ‘plain-language’ reports Multi Locus Sequence Typing + One Disruptive Technology Fits for Any Bacteria and Any Study Type aroE • Multi Locus Sequence Typi ng (MLST): 5-7 housekeeping genes distributed over the
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