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The Identification and Characterisation of Novel Antimicrobial Resistance

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The Identification and Characterisation of Novel Antimicrobial Resistance The identification and characterisation of novel antimicrobial resistance genes from human and animal metagenomes Liam Joseph Reynolds This thesis is submitted for the degree of PhD This PhD project was supervised by Dr. Adam P. Roberts: UCL Eastman Dental Institute, Department of Microbial Diseases and Dr. Muna F. Anjum: Animal and Plant Health Agency, Department of Bacteriology 1 Declaration I, Liam Joseph Reynolds, confirm that the work detailed in this thesis is my own except where stated otherwise. Saliva samples were collected from volunteers by Mr. Supathep Tansirichaiya (Eastman Dental Institute, UCL, United Kingdom). Calf faeces was collected by various members of staff at the Animal Plant and Health Agency. The creation of the human saliva and calf faecal 16S Illumina libraries was conducted in conjunction with Ms. Catryn Williams (Eastman Dental Institute, UCL, United Kingdom). Illumina MiSeq sequencing of these 16S libraries was carried out by Dr. Tony Brooks (Institute of Child Health, UCL, United Kingdom). End sequencing of a subset of the human saliva metagenomic library was conducted by myself and Mrs. Supanan Sucharit. A subset of the human saliva metagenomic library was screened by Mr. Gianmarco Cristarella under my supervision as part of his master’s research project. Analysis of the CTAB-resistant clone identified during this screen was completed in conjunction with Mr. Gianmarco Cristarella and Mr. Supathep Tansirichaiya. 2 Acknowledgements I would like to show my sincere gratitude to my supervisors Dr. Adam Roberts and Dr. Muna Anjum for the time, guidance and support they have given me over the last four years, as I was conducting laboratory work and writing this thesis. For the work they have conducted, that has contributed to my PhD, I would also like to thank Mr. Supathep Tansirichaiya, Ms. Catryn Williams and Mrs. Supanan Sucharit. Additionally, I would like to thank Dr. Anna Tymon, Mrs. Ingrid Green, Dr. Haitham Hussain, Dr. Franky Kaiser, Dr. Michael Brouwer, Dr. Roderick Card and Dr. Manal AbuOun for their technical guidance, particulary when I first started at the Eastman Dental Institute and the Animal and Plant Health Agency. In addition to their technical guidance and contributions, those who I have mentioned above have also become good friends and in this respect, I would also like to mention Ms. Marika Rai, Mr. Mehmet Davrandi, Mr. Ajijur Rahman and Ms Asyura Amdan. Throughout my PhD I have had continuous support and encouragement from my Mam and Dad, my three sisters, Danielle, Christine and Rebekah and my partner Lauren, for which I am immensely thankful. The work detailed in this thesis was jointly funded by a UCL IMPACT studentship (156780) and by the Seedcorn Programme at the Animal and Plant Health Agency (Seedcorn fund, SC1203). 3 Abstract Antimicrobial resistance genes are harboured by bacteria in the human oral cavity and ruminant faeces and they are shed in particularly high abundances in calf faeces. Furthermore, bacteriocin (antimicrobial peptide) producing bacteria have been isolated from these environments. In recent times bacteriocins have received much attention as potential alternatives to antibiotics. Human saliva and calf faeces harbour ‘yet-to-be cultured bacteria’ that can only be studied by analysing their DNA. To this end, two metagenomic libraries were created from human saliva and calf faeces metagenomic DNA with the aim of identifying novel antimicrobial resistance and bacteriocin genes. Screening these libraries for tetracycline resistance identified two tetracycline resistant clones. Clone PS9 was also tigecycline resistant and contained a 7,765 bp insert that encoded two half-ABC transporter genes; subcloning of these genes showed that they were responsible for the observed resistance phenotype. As the ABC transporter conferred resistance only to tetracyclines and its putative amino acid sequence showed <80 % identity to known tetracycline resistance proteins, it was named TetAB(60). Clone TT31 contained a 14,226 bp insert. 7, 216 bp of the insert had 97 % nucleotide identity to Tn916 and contained part of tet(M) and a full length tet(L) gene. This gene organisation has not been described in Tn916-like elements and it may represent a novel Tn916-like element. 4 The human saliva library was also screened for antiseptic resistance revealing a CTAB resistant clone. Random transposon mutagenesis of the 19.1 Kb insert and subcloning of a UDP-glucose 4-epimerase revealed it to be solely required for the observed resistance. This study identified novel tetracycline, tigecycline and CTAB resistance genes from the human saliva metagenome, demonstrating the importance of this environment as a source of resistance genes that may compromise the effectiveness of these antibiotics and antimicrobials. Additionally, this work highlights the relevance of house-keeping genes to the development of antimicrobial resistance. 5 Contents Title Page…………………………………………………………………………………………………………………….. 1 Declaration………………………………………………………………………………………………………………….. 2 Acknowledgements……………………………………………………………………………………………………… 3 Abstract..………………………………………………………………………………………………………………………4 Contents……………………………………………………………………………………………………………………….6 List of Figures………………………………………………………………………………………………………………. 18 List of Tables……………………………………………………………………………………………………………….. 22 Abbreviations………………………………………………………………………………………………………………. 24 Chapter 1 Introduction……………………………………………………………………. ……………………. 28 1.1 Antibiotics: Discovery………………………………………………………………… 29 1.1.1 Pre-Antibiotic Era………………………………………………………………………. 29 1.1.2 Discovery and Introduction of Antibiotics……………………………………30 1.1.2.1 Tetracyclines……………………………………………………………………………… 32 1.1.2.1.1 Discovery and Structure…………………………………………………………….. 32 1.1.2.1.2 Mode of Action………………………………………………………………………….. 33 1.1.2.1.3 Clinical Uses of Tetracyclines……………………………………………………… 37 1.1.2.1.4 Agricultural Uses of Tetracyclines………………………………………………. 37 1.1.2.1.5 Novel Tetracycline Derivatives…………………………………………………… 38 1.1.2.2 β-lactams…………………………………………………………………………………… 40 1.1.2.2.1 Structure and Diversity…………………………………………………………….... 40 1.1.2.2.2 Mode of Action………………………………………………………………………….. 42 6 1.1.2.2.3 Clinical Uses of β-lactams…………………………………………………………… 43 1.1.2.2.4 Agricultural uses of β-lactams……………………………………………………. 44 1.1.2.2.5 Novel β-lactam Therapies……………………………………………………………45 1.2 Antimicrobial Activity of Metals and Cationic Antiseptics………….. 45 1.2.1 Metals……………………………………………………………………………………….. 45 1.2.1.1 History of Metals as Antimicrobials……………………………………………. 45 1.2.1.2 Mode of Action of Copper and Silver Antimicrobial Activities…….. 46 1.2.1.3 Uses of Silver and Copper…………………………………………………………… 48 1.2.2 Cationic Antiseptics……………………………………………………………………. 48 1.2.2.1 Chlorhexidine…………………………………………………………………………….. 48 1.2.2.2 Quaternary Ammonium Compounds…………………………………………. 50 1.2.2.3 Uses of Cationic Antiseptics……………………………………………………….. 52 1.3 Antimicrobial Resistance……………………………………………………………. 52 1.3.1 The Increasing Threat of Antibiotic Resistance…………………………… 52 1.3.2 Intrinsic Resistance……………………………………………………………………. 55 1.3.3 Acquired Resistance…………………………………………………………………… 56 1.3.3.1 Mutation Contributes to Antimicrobial Resistance…………………….. 57 1.3.3.2 Horizontal Gene Transfer and Antimicrobial Resistance…………….. 58 1.3.3.2.1 Mechanisms of Horizontal Gene Transfer…………………………………… 58 1.3.3.2.1.1 Conjugation……………………………………………………………………………….. 58 1.3.3.2.1.1.1 Mobilisation of Non-Conjugative Elements………………………………… 60 1.3.3.2.1.2 Transformation………………………………………………………………………….. 61 1.3.3.2.1.3 Transduction……………………………………………………………………………… 63 7 1.3.4 Dissemination of Antimicrobial Resistance………………………………….64 1.3.5 Selection of Antimicrobial Resistance………………………………………….66 1.3.6 Resistance Can Persist in the Absence of Antibiotic Selection………69 1.3.7 Co-selection of Antibiotic Resistance Genes.………………………………. 70 1.3.8 Specific Mechanisms of Resistance…………………………………………….. 71 1.3.8.1 Tetracycline Resistance……………………………………………………………….71 1.3.8.1.1 Ribosomal Protection………………………………………………………………… 73 1.3.8.1.2 Tetracycline Efflux……………………………………………………………………….75 1.3.8.1.3 Enzymes and Tetracycline Resistance…………………………………………. 76 1.3.8.1.4 Further Mechanisms of Resistance………………………………………………77 1.3.8.2 β-lactam Resistance…………………………………………………………………….78 1.3.8.2.1 β-lactamases……………………………………………………………………………….79 1.3.8.2.1.1 Classification of β-lactamases…………………………………………………….. 79 1.3.8.2.1.2 β-lactamase Mechanisms of Hydrolysis……………………………………….81 1.3.8.2.2 Penicillin Binding Protein Affinity………………………………………………..83 1.3.8.3 Metal Resistance…………………………………………………………………………85 1.3.8.4 Reduced Susceptibility to Cationic Antiseptics……………………………. 88 1.3.9 The Importance of Antimicrobial Resistance Research……………….. 91 1.4 Bacteriocins As Alternatives to Antibiotics…………………………………. 91 1.4.1 Classification of Bacteriocins……………………………………………………….92 1.4.2 Molecular Biology of Bacteriocins………………………………………………. 94 1.4.3 Mode of Action of Bacteriocins……………………………………………………98 1.4.3.1 Inhibition by Lipid II Binding………………………………………………………..98 8 1.4.3.2 Pore-Forming Bacteriocins…………………………………………………………. 100 1.4.3.3 Enzymatic Bacteriocins………………………………………………………………. 102 1.4.3.4 Bacteriocins Targeting DNA, RNA and Protein Synthesis…………….. 102 1.4.4 Clinical Applications of Bacteriocins…………………………………………… 103 1.4.5 Resistance to Bacteriocins………………………………………………………….. 104 1.4.6 The Importance of Identifying Novel Bacteriocins………………………. 105 1.5 Metagenomics…………………………………………………………………………… 106 1.5.1 The Beginning of Metagenomics…………………………………………………
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