Characterization of methicillin-resistant Staphylococcus aureus Sequence Type 398 Mette Theilgaard Christiansen PhD Thesis 2014 Characterization of methicillin-resistant S taphylococcus aureus Sequence Type 398 PhD thesis by Mette Theilgaard Christiansen, cand.scient.techn. Revised version Submitted February 2014 National Food Institute Technical University of Denmark Division for Epidemiology and Microbial Genomics Kemitorvet building 204, 2800 Kgs. Lyngby, DK Preface The thesis is a result of a three-year PhD project carried out at the national Food Institute at the Technical University of Denmark in the Division of Epidemiology and Microbial Genomics. The project was supervised by Professor Frank Møller Aarestrup as main supervisor and Senior Researchers Henrik Hasman and Yvonne Agersø as co-supervisors. The project was conducted from June 2010 to August 2013 and a six month external research stay was included from October 2011 to March 2012 at Department of Veterinary Medicine, University of Cambridge, UK under the supervision of Senior Lecturer Dr. Mark Holmes. The work was funded by the Danish Ministry of Food, Agriculture and Fisheries (Grant no.: 3304-FVFP-09-F-002-1) and The Technical University of Denmark. The thesis consists of an introduction and three manuscripts all presented in three chapters. Chapter 1 includes a short introduction to Staphylococcus aureus and to some of the methods used for bacterial characterization. Studies characterizing S. aureus and especially S. aureus multilocus sequence type 398 (ST398) are presented. In chapter 2 the results obtained in the three manuscripts are summarized and discussed. Chapter 3 contains the three manuscripts (manuscript I-III). Manuscript I describes the generation, verification, and evaluation of a high-throughput approach for bacterial characterization. In manuscript II a genomic screen was performed to identify genes important for S. aureus ST398 survival in a porcine reservoir. Manuscript III represents the development of a bioinformatic tool that can be used for virulence profiling of S. aureus using whole genome sequence data. 1 Supervisors: Professor Frank M. Aarestrup (main supervisor), Research Group of Microbial Genomics and Antimicrobial Resistance, Division of Epidemiology and Microbial Genomics, National food ‘institute, Technical University of Denmark Senior Scientist Henrik Hasman (co-supervisor), Research Group of Microbial Genomics and Antimicrobial Resistance, Division of Epidemiology and Microbial Genomics, National food ‘institute, Technical University of Denmark Senior Scientist Yvonne Agersø (co-supervisor), Research Group of Microbial Genomics and Antimicrobial Resistance, Division of Epidemiology and Microbial Genomics, National food ‘institute, Technical University of Denmark Assessment committee: Senior Scientist Lina Cavaco , Research Group of Microbial Genomics and Antimicrobial Resistance, Division of Epidemiology and Microbial Genomics, National food ‘institute, Technical University of Denmark Professor Jody A. Lindsay , Research Centre for Infection and Immunity, Division of Clinical Sciences, St. George’s University of London, UK Professor Henrik Westh , Department of Clinical Microbiology, Hvidovre Hospital, DK 2 Acknowledgements There are a lot of people I want to thank, who in one way or another have contributed to the completion of this work. First of all I would like to thank my supervisor Frank Aarestrup for the last three years. It has been hard, fun, frustrating, challenging but most of all educational. Also thank you Henrik and Yvonne for your co-supervision and for, at times, putting Frank’s ideas into a more realistic context. Another very important person I would like to thank is Mark Holmes, my external supervisor at Cambridge University. You opened up your lab for me without hesitation. You and your colleagues helped me through a though period where no experiments would work and you have opened a door of opportunity in the UK for me – thank you! Acknowledgement and many thanks to all my colleagues at DTU Food! You have definitely made my stay at DTU a pleasant one and without your assistance I would have been lost. Thanks to Maria and Rita for being the best officemates one could ever hope for. You have been great in every aspect. A special thanks goes to Rolf! You have helped me tremendously with my data analysis and you have contributed to my bioinformatic skills in a way that I’m now not afraid to take a job using bioinformatics on a daily basis. I would also like to thank all my wonderful friends for putting up with me and my talk about MRSA the past years. It must have been tiring at times, but also very exciting, right? Anne, thank you so much for proof reading everything, it has really been a great help. Thank you to my family, you’re great and I love you. Mom and dad, thank you for always being supportive and proud of me no matter what! Above all, thanks to my amazing husband. Kenneth, you have been more than any, the greatest support, suffering with me at times and celebrating at others. Your high expectations continue to push me and you make me feel like I can do anything. Thank you! 3 English summary Staphylococcus aureus is an opportunistic pathogen that colonizes the nares and skin surfaces of several animal species, including man. S. aureus can cause a wide variety of infections ranging from superficial soft tissue and skin infections to severe and deadly systemic infections. Traditionally S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) have been associated with hospitals, but during the past decades MRSA has emerged in the community and now a new branch of MRSA has been found in association with livestock (LA-MRSA). A specific lineage (multilocus sequence type 398 (ST398)) has been particularly successful in colonization of pigs and ST398 has become the most frequently reported MRSA strain found in associated with livestock. Currently the understanding of the successful colonization and transmission of LA-MRSA ST398 in pigs are limited and mainly based on observational field surveys. The aim of this work was to develop a high-throughput approach for genotypic and phenotypic characterization of LA-MRSA ST398 in the porcine reservoir. The thesis represents three studies (manuscript I-III). In manuscript I a genome-saturated transposon mutant library was generated and Transposon Directed Inserted site Sequencing (TraDIS) was for the first time assessed in an LA-MRSA ST398 strain. Using this high-throughput approach, genes essential for LA-MRSA ST398 survival under laboratory conditions and in whole porcine blood in vitro were identified. In manuscript II, genes important for LA-MRSA ST398 survival on porcine skin and nasal epithelium ex vivo were identified. These genes could represent targets for de-colonization, which could help prevent further spread and adaption of LA-MRSA ST398. Manuscript III describes the construction of the S. aureus VirulenceFinder database. The database can be applied for identification of virulence genes in S. aureus using whole genome 4 sequence data. The S. aureus VirulenceFinder will be part of the tool package generated for the Centre for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). 5 Dansk resumé Staphylococcus aureus er en opportunistisk patogen, der koloniserer næsen og huden hos forskellige dyrearter, inklusive mennesker. S. aureus kan forårsage en lang række forskellige infektioner, der bl.a. omfatter overfladiske hudinfektioner og mere alvorlige systemiske infektioner. Traditionelt har S. aureus og methicillin-resistente Staphylococcus aureus (MRSA) været associeret med hospitalserhvervet infektioner, men i de seneste årtier har MRSA spredt sig til resten af samfundet, og en ny gren af MRSA med association til produktionsdyr er blevet identificeret (LA- MRSA). En specifik slægt har vist sig at være yderst succesfuld til at kolonisere grise, og denne slægt (ST398) er nu den hyppigst rapporterede MRSA stamme fundet i association med produktionsdyr. Den nuværende viden, om hvorfor LA-MRSA ST398 er succesfuld i kolonisation og spredning hos grise, er begrænset og primært baseret på overvågnings-studier. Formålet med dette studie var at udvikle en metode til at udføre en omfattende genotypisk og fænotypisk karaktering af LA-MRSA ST398 i et grise-reservoir. Denne afhandling repræsenterer tre studier (manuskript I-III). I manuskript I blev der genereret et genom-mættet transposon mutant bibliotek, og ’Transposon Directed Inserted site Sequencing (TraDIS)’ blev for første gang anvendt på en LA-MRSA ST398 stamme. Ved brugen af disse metoder blev gener essentielle for LA-MRSA ST398s overlevelse under laboratorie forhold og i svine blod identificeret. I manuskript II blev gener vigtige for LA-MRSA ST398 overlevelse på svine hud og svine-næseepitel bestemt. Disse gener kan potentielt udgøre nye mål for afkolonisering og dermed forebygge videre spredning og tilpasning af LA-MRSA ST398. Manuskript III beskriver konstruktionen af en S. aureus VirulenceFinder database. Denne kan anvendes til identifikation af virulens gener i S. aureus hel-genom sekvens data. S. aureus 6 VirulenceFinder er en del af den redskabspakke, der bliver genereret for Center for Genomisk Epidemiologi (CGE) (www.genomicepidemiology.org). 7 Table of Contents Preface .............................................................................................................................................................. 1 Acknowledgements .........................................................................................................................................