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Identification of Genes Involved in Acid Tolerance, Antimicrobial Resistance and Virulence of Enterococcus Faecium

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Identification of Genes Involved in Acid Tolerance, Antimicrobial Resistance and Virulence of Enterococcus Faecium Identification of genes involved in acid tolerance, antimicrobial resistance and virulence of Enterococcus faecium Thesis submitted by Hashimatul Fatma binti Hashim For the degree of DOCTOR OF PHILOSOPHY in the Faculty of Medical Sciences University College London Division of Microbial Diseases UCL Eastman Dental Institute 256 Gray’s Inn Road London WC1X 8LD UK 2016 Declaration I hereby certify that the work embodied in this thesis is the result of my own investigation, except where otherwise stated. 2 Abstract Enterococcus faecium is an opportunistic human nosocomial pathogen that has developed resistance to many existing antimicrobial therapies. Treatment of enterococcal infections is becoming increasingly challenging and there are limited therapeutic options against multidrug-resistant enterococci. The work described in this thesis aimed to identify genes involved in resistance to host stresses and virulence as these could potentially be future therapeutic targets. An E. faecium transposon mutant library was screened for altered resistance to lysozyme and to nisin as a model antimicrobial peptide. This approach led to the identification of several genes that contribute to lysozyme and nisin resistance. A number of mutants that were sensitive to nisin had a transposon insertion in a gene predicted to encode a tyrosine decarboxylase. It was shown that the tyrosine decarboxylase plays a role in acid tolerance and mediates virulence of E. faecium in a Galleria mellonella larvae infection model. A targeted genetic approach was used to examine the role of an E. faecium serine threonine protein kinase (Stk1) in antimicrobials resistance, host cell stresses and virulence. Disruption of the stk1 gene led to higher sensitivity to antibiotics that target the penicillin- binding proteins and bile salts compared to the wild type. Providing the stk1 gene from either E. faecium or ireK of E. faecalis in trans restored the resistance of the E. faecium stk1 mutant to ceftriaxone. The stk1 gene also contributed to E. faecium virulence in the G. mellonella infection model. Through bioinformatic analysis a putative ATP-binding-cassette (ABC) transporter system with the potential to be involved in antimicrobial peptide resistance was identified. Markerless deletion of the gene encoding the permease component of the ABC transporter reduced E. faecium resistance to the antimicrobial peptide nisin and altered the net charge of the bacterial cell surface. Genes involved in acid tolerance, antimicrobial resistance and virulence of E. faecium were identified in this study. 3 Table of Contents Declaration................................................................................................................................2 Abstract.....................................................................................................................................3 Table of Contents......................................................................................................................4 List of Figures............................................................................................................................8 List of Tables............................................................................................................................13 Abbreviations..........................................................................................................................15 1 General Introduction ....................................................................................................... 17 1.1 Enterococcus faecium ................................................................................................ 17 1.2 Enterococci ................................................................................................................ 17 1.3 Infections caused by Enterococci .............................................................................. 18 1.4 Emergence of Clonal Complex 17 Enterococcus faecium ......................................... 20 1.5 Emergence of antibiotic resistance enterococci ....................................................... 22 1.5.1 Bile salts resistance ............................................................................................ 26 1.6 Enterococcal cell wall ................................................................................................ 27 1.6.1 Peptidoglycan ..................................................................................................... 27 1.6.2 Wall teichoic acids and lipothechoic acids ........................................................ 28 1.6.3 Cell wall-anchored proteins ............................................................................... 30 1.7 Virulence-associated factors in E. faecium ............................................................... 31 1.7.1 Esp ...................................................................................................................... 31 1.7.2 HylEfm ................................................................................................................ 33 1.7.3 SagA .................................................................................................................... 34 1.7.4 Acm .................................................................................................................... 35 1.7.5 Scm ..................................................................................................................... 35 1.7.6 SgrA .................................................................................................................... 36 1.7.7 EcbA ................................................................................................................... 36 1.7.8 Pili ....................................................................................................................... 37 1.7.9 AsrR .................................................................................................................... 38 1.7.10 GelE .................................................................................................................. 40 1.8 Aim of this project ..................................................................................................... 40 2 Materials and methods ..................................................................................................... 43 4 2.1 Bacterial strains ......................................................................................................... 43 2.2 Plasmids ..................................................................................................................... 45 2.3 Media ........................................................................................................................ 46 2.4 Growth conditions ..................................................................................................... 46 2.5 Antibiotics.................................................................................................................. 47 2.6 Molecular biology techniques ................................................................................... 47 2.6.1 Plasmid DNA purification ................................................................................... 47 2.6.2 Genomic DNA purification ................................................................................. 47 2.6.3 Agarose gel electrophoresis ............................................................................... 47 2.6.4 Standard PCR protocol ....................................................................................... 48 2.6.5 Colony PCR ......................................................................................................... 50 2.6.6 Lyse and go reaction .......................................................................................... 50 2.6.7 DNA purification from gel .................................................................................. 50 2.6.8 Restriction endonuclease reactions ................................................................... 51 2.6.9 Ligation ............................................................................................................... 51 2.6.10 Dephosphorylation reactions ........................................................................... 51 2.6.11 5’-Phosphorylation of DNA fragment ............................................................... 51 2.7 DNA Sequencing ........................................................................................................ 52 2.8 The pHOU1 counter selection system....................................................................... 52 2.9 Preparation of competent cells ................................................................................. 53 2.9.1 Preparation of electro-competent E. faecium and E. faecalis ........................... 53 2.9.2 Preparation of competent cells of E. coli EC1000 .............................................. 53 2.10 Transformation .................................................................................................. 54 2.10.1 Transformation of E. faecium and E. faecalis ................................................... 54 2.10.2 Transformation of E. coli ................................................................................... 54 2.11 Blue/white colour screening ..................................................................................... 55 2.12 Determination of bacterial
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