16S rRNA methyltransferases: the end of aminoglycosides? Emma Louise Taylor Imperial College London Department of Medicine PhD in Clinical Medicine Research 2020 1 Declaration of originality I, Emma Louise Taylor, confirm that the work presented in this thesis is entirely my own, and if otherwise, is referenced accordingly. Copyright declaration The copyright of this thesis rests with the author. Unless otherwise indicated, its contents are licensed under a Creative Commons Attribution-Non Commercial 4.0 International Licence (CC BY-NC). Under this licence, you may copy and redistribute the material in any medium or format. You may also create and distribute modified versions of the work. This is on the condition that: you credit the author and do not use it, or any derivative works, for a commercial purpose. When reusing or sharing this work, ensure you make the licence terms clear to others by naming the licence and linking to the licence text. Where a work has been adapted, you should indicate that the work has been changed and describe those changes. Please seek permission from the copyright holder for uses of this work that are not included in this licence or permitted under UK Copyright Law. 2 Abstract 16S rRNA methyltransferases (16S RMTases) confer high-level aminoglycoside resistance (MICs >256 mg/L) to Gram-negative bacteria and are an emerging threat. The prevalence of 16S RMTases in the UK is entirely unknown as are the drivers behind their emergence. The occurrence of 16S RMTases (ArmA, RmtA-RmtH and NpmA) in the UK was identified by screening bacteria from Public Health England’s Antimicrobial Resistance and Healthcare Associated Infections Reference Unit from 2003-2015 and whole-genome sequences from the British Society for Antimicrobial Chemotherapy Resistance Surveillance Project from 2001- 2013, with positivity rates of 83.2% (1,312/1,576) and 0.3% (4/1,566), respectively. A prospective surveillance study, where 14 NHS Trusts sent amikacin-resistant bacterial isolates from May 1st to October 31st 2016, determined a period prevalence of 16S RMTases of 0.1% (79/71,063). Potential risk factors for acquisition of 16S RMTase-producing bacteria, were identified using anonymised case questionnaires and included age (≥65 years), being male, an inpatient or a non-UK resident and receiving medical treatment abroad. 16S RMTases were frequently associated with carbapenemases, which were identified in 94.3% (1,237/1,312), 87.3% (69/79) and 50.0% (2/4) 16S RMTase-producing isolates in these isolate collections. 16S RMTases were frequently carried by ‘high-risk’ bacterial clones such as Klebsiella pneumoniae ST14. Analysis of genome sequence data identified mobile genetic elements such as Tn1548 (armA), Tn2 (rmtB), ISEcp1 (rmtC) and IS91 (rmtC and rmtF) were associated with 16S RMTases. Analysis of plasmids identified genetic linkage of 16S RMTases with the carbapenemase NDM-1 and the circulation of novel plasmids within the UK. Although currently rare in the UK, 16S RMTases appear to be emerging through clonal expansion, and potentially through association with carbapenemases and mobile genetic elements. Given the critical therapeutic role of aminoglycosides in combatting the challenge of antimicrobial resistance in Gram negative pathogens, the risk of future emergence is high, underlining a need for ongoing surveillance. 3 Acknowledgements Firstly, I would like to thank the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Healthcare Associated Infections and Antimicrobial Resistance at Imperial College London in partnership with Public Health England (PHE) for funding my PhD project. Special thanks to my supervisors Dr Katie Hopkins, Professor Neil Woodford and Professor Shiranee Sriskandan for supporting me throughout my PhD and offering their invaluable advice to help me write my thesis and help me through my assessments and conference presentations. I want to thank Claire Perry, Amy Coward, Alice Fuller and Noshin Sajedi from the AMRHAI Reference Unit at PHE for their assistance with strain characterization, especially Dr Jane Turton for going above and beyond to help me whenever I was in need. Your kindness and support was very much appreciated. Many thanks go to Dr Rachel Pike, Aiysha Chaudry and Rachael Adkin from the AMRHAI Reference Unit for their help with antibiotic susceptibility testing; Daniel Godoy and Nazim Mustafa from the AMRHAI Reference Unit for their help with carbapenemase screening and Neville Verlander from the Statistics, Modelling and Economics Department at PHE for guidance during the statistical analysis. Special thanks to all of the laboratory staff and Consultant Microbiologists who participated in the prospective surveillance study and Professor Alan Johnson at PHE and Dr Michael Lockhart from Health Protection Scotland for their help with hospital recruitment. I would also like to thank Professor Julian Parkhill and Professor Sharon Peacock for allowing me to work at the Wellcome Trust Sanger Institute and for giving me access to data from the British Society for Antimicrobial Chemotherapy Resistance Surveillance Project. Additional thanks go to the Wellcome Trust Sanger Institute for carrying out Illumina sequencing on the bacterial isolates from this project. 4 Many thanks to Professor Bruno González-Zorn, Dr Laurent Poirel and Dr Yohei Doi for providing bacterial positive controls for rmtA + rmtE + rmtF, rmtG and rmtH, respectively. I’m extremely grateful to Dr Elita Jauneikaite for supporting me throughout my PhD and for teaching me how to use bioinformatics software. Thank you for encouraging me when times were tough and for always being there for me when I was in need. Thank you to all of my family and friends for their love and support throughout the last four years. 5 Table of contents Declaration of originality ........................................................................................................ 2 Copyright declaration ............................................................................................................ 2 Abstract................................................................................................................................. 3 Acknowledgements ............................................................................................................... 4 Figures ................................................................................................................................ 14 Tables ................................................................................................................................. 17 Abbreviations ...................................................................................................................... 21 Chapter 1: Introduction ........................................................................................................ 24 1.1 Antibiotic resistance .............................................................................................. 25 1.1.1 Discovery of antibiotics........................................................................................ 25 1.1.2 Antibiotic resistance ............................................................................................ 25 1.1.3 Microbiological investigation of antibiotic-resistant bacteria ................................. 30 1.1.3.1 Antibiotic susceptibility testing ...................................................................... 30 1.1.3.2 Bacterial typing ............................................................................................. 33 1.1.3.2.1 PFGE ..................................................................................................... 33 1.1.3.2.2 VNTR analysis ....................................................................................... 33 1.1.3.2.3 MLST ..................................................................................................... 34 1.1.3.2.4 WGS ...................................................................................................... 36 1.1.3.2.5 Uses of bacterial typing .......................................................................... 36 1.1.4 Concern regarding antibiotic resistance in Gram-negative bacteria ..................... 36 1.2 Aminoglycosides ........................................................................................................ 37 1.2.1 Discovery of aminoglycosides ............................................................................. 37 1.2.2 Chemical structure .............................................................................................. 39 1.2.3 Advantages and disadvantages of aminoglycoside use....................................... 39 1.2.4 Toxicity ................................................................................................................ 41 1.2.5 The use of aminoglycosides in the UK ................................................................ 41 1.2.6 Uptake and mechanism of action ........................................................................ 42 1.2.6.1 Initial uptake ................................................................................................. 42 1.2.6.2 Energy-dependent phases I and II ................................................................ 43 1.2.7 Rates of aminoglycoside resistance in the UK ..................................................... 45 1.2.8 Mechanisms of aminoglycoside resistance.........................................................