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

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

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

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

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

ESCMID Online Lecture Library

Mellmann et al. (2011). PLoS One. 6: e22751 [PubMed] Timeline – Microbial Surveillance and Disease

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

+ 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) distributed 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

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 whole genome. One single expanding nomenclature. • Used mainly for population genetics and evolutionary studies. + gdh • MLST : genome-wide gene by gene typing; pdhC hundreds/thousands of pgm, adk core genome genes distributed over the whole genome. Central public abcZ © by author global nomenclature server (‘Molecular Ty ping Esperanto’) essential. MaidenESCMID et al. (1998). PNAS 95: 3140 Online [PubMed]. Lecture• LibraryDue to high discriminatory power also suited for outbreak investigations.

Combine the Best - Comparability of MLST and discriminatory power of PFGE MLST+ - Software Tools

/ Ridom SeqSphere+ • BIGSdb

Jolley & Maiden (2010). BIGSdb. BMC Bioinformatics 11: 595 [PubMed], Jünemann et al. (2013). Nature Biotechnology 31(4): 294 [PubMed]. Hierarchical Microbial Typing Approach

Lineage specific SNPs / e.g., Köser et al (2012). NEJM 366: 2267 [PubMed] Alleles explorative Standardized

Species specific

Mellmann et al. (2011). PLoS One. 6: e22751 [PubMed] + MLST Vogel et al. (2012). JCM 50: 1889 [PubMed] Jolley et al. (2012). JCM. 50: 3046 [PubMed]

Species specific SNPs e.g., Van Ert et al. (2007). JCM 45: 47 [PubMed] ESCMIDconfirmatory/canonica Online l Lecture Library Maiden et al. (1998). PNAS 95: 3140 [PubMed] MLST also needed for backward compatibility Hierarchical microbial typing approach. From bottom to top with i ncreasing discriminatory power. M L S T, multi locus sequence typing ; r MLS T, ribosomal MLST; SNP, single nucleotide polymorphism.

*user must register (free for academicians) at http://pubmlst.org/rmlst Initiatives and Projects

http://www.globalmicrobialidentifier.org/

Aarestrup et al. (2012). EID 18: e1. doi: 10.3201 [PubMed] European Projects Center for Genomic Epidemiology

5 Mio. €, 2010-2016, Danish Council for Strategic Research (09-067103/DSF)

ESCMID Online Lecturehttp://www.modmedmicro.ac.uk/ Library

6 Mio. €, 2012-2016, European Community‘s Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. 278864

http://patho-ngen-trace.eu/ © by author ESCMID Online Lecture Library © by author • Research Center Borstel, Leibniz Center for Biomedical Research, Germany (Stefan Niemann; coordinator) • Westfälische Wilhelms-Universität Münster, Germany (Dag Harmsen) • University of Oxford, Department of Zoology, United Kingdom (Martin Maiden)

• RidomESCMID GmbH, Germany (Jörg Rothgänger)Online Lecture Library • Applied Maths NV, Belgium (Bruno Pot) • Genoscreen SAS, France (Caroline Allix-Béguec) • Piext BV, Netherlands (Ronald Burggrave) • European Research and Project Office GmbH, Saarbrücken, Germany (Claudia Schacht) Outlook

• whole genome microbial typing

• transmission chain analysis & GIS

• resistome and pathogenome/toxome analysis

• plain language report

• unique signature detection© by for author development of rapid molecular screening tests ESCMID Online Lecture Library Transmission Chains from Four Dimensions

Illustration of possible reconstructions© of genealogicalby author relations. Panel a represents a hypothetical genealogy of six isolates from which three were sampled (highlighted by a blue glow and numbered from 1 to 3). Panels b and c illustrate phylogenetic reconstructions where the red dots represent inferred nodes; panel b corresponds to a classical phylogenetic reconstruction and c to a tree that accountsESCMID for heterochronous samplingOnline (e.g., BEAST Lecture software). Panel Library d illustrates the direct ancestry reconstruction method implemented in SeqTrack.

Jombart et al. (2011). Heredity. 106: 383 [PubMed] EpiScanGis Meningococcal infectious disease in Germany • creation of a web- based application • user-friendly interface • allowing queries as to the distribution of fine types, incidence rates etc. • since 2006 weekly prospective scans with SaTScan, display and storage of results • used by 50% of all © by author local German public health authorities for informedESCMID decisions Online Lecture Library (vaccination cam-

paigns) Elias et al. (2006). Emerg. Infect. Dis. 12: 1689 [PubMed], http://www.EpiScanGis.org/ Reinhardt et al. (2008). Int. J. Health Geogr. 7: 33 [PubMed]. Grundmann et al. (2010). PLoS Med 7: [PubMed].

Köser et al (2012). NEJM 366: 2267 [PubMed]

Dag Harmsen University of Münster, Germany