Kingston University London The Antibiotic Resistance Growth Plate (ARGP) as an experimental evolution tool to explore the phenotypic and genotypic mutational pathways underlying the emergence of antimicrobial resistance in Escherichia coli. A thesis submitted in partial fulfilment for the degree of Doctor of Philosophy By Lucky Bonnie Lucia CULLEN Faculty of Science, Engineering and Computing February 2019 Declaration This thesis entitled ‘The Antibiotic Resistance Growth Plate (ARGP)’ as an experimental evolution tool to explore the phenotypic and genotypic mutational pathways underlying the emergence of antimicrobial resistance in Escherichia coli’ is based upon the work conducted in the Faculty of Science, Engineering and Computing at Kingston University London and in collaboration with Dr Philip Aldridge at Newcastle University and Katie Hopkins and Neil Woodford from Public Health England. All the work described is the candidate’s own original work unless otherwise stated. None of the work presented has been submitted for another degree internally or externally. i Acknowledgements At the start of this PhD I had a dream, a dream which could have never been accomplished without the support of many people. Firstly, I would like to express my sincere appreciation to my director of studies Professor Mark Fielder, your provision, guidance and unconditional belief in my potential has contributed significantly to the completion of this thesis. I would also like to acknowledge the overwhelming support received from, Dr Scott Lawton, Camilla Eldridge and Ben Jones, your wealth of knowledge and expertise in the field of evolutionary biology and bioinformatics has assisted greatly in concluding the work within this PhD. I would also like to praise the incredible multidisciplinary support received at Kingston University, specifically that from Dr Gary-Forster Wilkins, Dr Adam Le Gresley, Dr Brian Rooney, Dr James Denholm-Price and Richard Giddens. I would also like to extend my gratitude to all those in the Microbiology laboratory including my lovely friends and technicians, who have made this experience enjoyable. A special thank you goes to Coral Brazier, from the Kingston University wellbeing team. Finally, I would like to thank my wonderful family and friends who have believed in me endlessly, thank you all for being a part of my journey. To my sister Kaycee thank you for being my glimmer of sunshine during the darkest times. The greatest thank you goes to my mum, nobody knows how challenging this journey has been like you do and neither would this have been possible without you. Everything, I am and everything I aspire to be, I owe to you. Nanny Kathy although you will not remember, I dedicate this to you. ii Publications Antibiotic Resistance Growth Plate (ARGP): A simple tool for exploring the evolution of antimicrobial resistance. Cullen LBL, Eldridge C, Forster-Wilkins G and Fielder MD (Article in preparation). Presentations Developing an experimental tool to explore the evolution of antimicrobial resistance? Oral presentation. Society for Applied Microbiology ECS Research Conference. The Royal Society of Medicine. October 2016. Exploring the evolutionary pathways to antimicrobial resistance using experimental evolution. Oral presentation. Society for Applied Microbiology Antimicrobial Resistance Conference. One Great George Street. November 2016. Characterising the emergence of antibiotic resistance through mutations using experimental evolution. Poster presentation. Science Engineering and Computing Conference. Kingston University London. April 2017. Characterising the emergence of antibiotic resistance: mutation mapping of the resistome. Oral presentation. American Society for Microbiology (ASM) Microbe Conference. New Orleans. June 2017. An alternative approach to tackling the antibiotic resistance crisis. Oral presentation. Three Minute Thesis winner and national semi-finalist. Kingston University London. 2017. iii List of Acronyms ° Degree °C Degrees Centigrade μg Micrograms μl Microliter μM Micromolar Nucleotide Diversity 3’ 3 prime end 5’ 5 prime end ABU Asymptomatic Bacteriuria AIC Akaike Information criterion ALE Adaptive Laboratory Evolution AACs Aminoglycoside Acetyltransferases AMEs Aminoglycoside Modifying Enzymes AMR Antimicrobial Resistance APHA Animal and Plant Health Agency ARGP Antibiotic Resistance Growth Plate ASM American Society for Microbiology BIC Bayesian Information Criterion BSAC British Society for Antimicrobial Chemotherapy CDC Centre for Disease Control and Prevention CDS Coding Sequence CFU/mL Colony Forming Units Per mL iv CLED Cysteine-Lactose-Electrolyte Deficient CPE Carbapenemase Producing Enterobacteria CRE Carbapenem-Resistant Enterobacteriaceae Cryo-EM Cryo-Electron Microscopy D2O Deuterium Oxide dH2O Distilled Water DMAB Dimethylaminobenzaldehyde dN/dS Non-synonymous/Synonymous dNTPs Fluorescently Tagged Nucleotides DT Decision Theory EARS-Net European Antimicrobial Resistance Surveillance Network E. coli Escherichia coli EDTA Ethylenediaminetetraacetic Acid EF-G Elongation Factor-G EF-Tu Elongation Factor-Tu EMBL-EBI European Bioinformatics Institute EPEC Enteropathogenic E. coli EUCAST European Committee on Antimicrobial Susceptibility Testing ExPEC Extraintestinal Pathogenic E. coli G. mellonella Galleria mellonella GF-Score Goodness-of-Fit Score GMQE Global Model Quality Estimate GTP Guanosine Triphosphate HGT Horizontal Gene Transfer v -InL Likelihood ICL Imperial College London iNTPs Initiating Nucleoside Triphosphate IS Insertion Elements iTOL Interactive Tree of Life KU Kingston University LCBs Locally Collinear Blocks LC-MS Liquid Chromatography- Mass Spectroscopy MEA Maximum Expected Accuracy MEGA Molecular Evolutionary Genetics Analysis MFE Minimum Free Energy MH Mueller Hinton MIC Minimum Inhibitory Concentration ML Maximum Likelihood MLST Multilocus Sequence Typing mRNA Messenger Ribonucleic Acid MSW Mutant Selection Window MUSCLE Multiple Sequence Comparison Log-Expectation MVA Multivariate Analyses NCBI National Centre for Biotechnology NCTC National Collection of Type Cultures NJ Neighbour Joining NMR Nuclear Magnetic Resonance NNI Nearest-neighbour Interchange vi nt Nucleotide NU Newcastle University OD Optical Density O/N Overnight OPG Osmoregulated Periplasmic Glucan OPLS-SA Orthogonal Partial Least Squares- Discriminant Analysis P. mirabilis Proteus mirabilis PBS Phosphate Buffered Saline PCA Principal Component Analysis PCR Polymerase Chain Reaction PDB Protein Data Bank PHE Public Health England ppGpp Guanosine5’-diphosphate 3’-diphosphate QMEAN Quality Model Energy QSQE Quaternary Structure Quality Estimate RND Resistance-Nodulation-Division rrn Ribosomal RNA rRNA Ribosomal Ribonucleic Acid SAPS Statistical Analysis of Protein Sequences SEM Scanning Electron Microscopy SfAM Society for Applied Microbiology SMART Simple Modular Architecture Research Tool smFRET Single-Molecule Fluorescence Resonance Energy Transfer SNP Single Nucleotide Polymorphism vii Spp. Species TBE Tris-Borate-EDTA TDR Totally Drug-Resistant TMS Tetramethylsilane trGTPases Translational GTPases TTC Triphenyltetrazolium Chloride VSEPR Valence Shell Electron Pair Repulsion WGS Whole Genome Sequencing WT Wild-Type viii Table of Contents List of acronyms ............................................................................................. iv Table of contents ............................................................................................ ix Table of figures ............................................................................................... xx Table of tables ................................................................................................. xxx Abstract……………………………………………………………………………xxxiii Chapter 1: Introduction .................................................................................. 1 1.1 Antimicrobial resistance ............................................................................. 1 1.1.1 The antimicrobial resistance threat .................................................... 1 1.1.2 Antimicrobial resistance: a one health perspective ............................ 2 1.1.3 Mechanisms of antimicrobial resistance ............................................. 3 1.1.3.1 Intrinsic mechanisms of antimicrobial resistance ..................... 4 1.1.3.2 Acquired mechanisms of antimicrobial resistance..................... 5 1.1.3.3 Acquired antimicrobial resistance through mutations ................ 5 1.1.4 The significance of Gram-negative bacteria ....................................... 7 1.1.4.1 Escherichia coli and its application as a model organism ......... 8 1.1.4.2 The importance of aminoglycoside antibiotics .......................... 11 1.2 The history of microbial culture methods ................................................... 12 1.2.1 The application of adaptive laboratory evolution (ALE) in microbiology. .......................................................................................................................... 12 1.2.2 Experimental approaches in the study of antimicrobial resistance ..... 13 1.2.3 The future of ALE studies ................................................................... 17 1.3 Fitness costs associated with resistance development .............................. 19 1.4 Phylogenetic interpretation of ALE findings ............................................... 20 1.5 Treatment strategies informed by ALE ....................................................... 20 1.6 Aims and objectives