Serratia marcescens: Outer Membrane Porins and Comparative Genomics By Alexander Diamandas A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements of the degree of Master of Science Department of Microbiology University of Manitoba Winnipeg Copyright © 2019 by Alexander Diamandas Table of Contents Abstract ........................................................................................................................................................ v Acknowledgments ...................................................................................................................................... vi List of Tables .............................................................................................................................................. vii List of Figures ............................................................................................................................................ viii 1. Literature Review: ............................................................................................................................ 10 1.1. Serratia marcescens .................................................................................................................... 10 1.1.1. Species ................................................................................................................................ 11 1.1.2. Incidence and Mortality ...................................................................................................... 15 1.1.3. Virulence and Pathogenesis in Humans .............................................................................. 16 1.2. Porins .......................................................................................................................................... 17 1.2.1. Background ......................................................................................................................... 17 1.2.2. Porins in S. marcescens ....................................................................................................... 18 1.2.2.1. OmpF .......................................................................................................................... 18 1.2.2.2. OmpC .......................................................................................................................... 19 1.2.2.3. PhoE ............................................................................................................................ 20 1.2.2.4. LamB ........................................................................................................................... 20 1.2.2.5. OmpA .......................................................................................................................... 21 1.2.2.6. OmpX .......................................................................................................................... 22 1.2.3. Assembly ............................................................................................................................. 23 1.3. S. marcescens and Antimicrobials .............................................................................................. 24 1.3.1. Antibiotics used in S. marcescens Treatment ..................................................................... 24 1.3.2. Drug Inactivating Enzymes ................................................................................................. 25 1.3.2.1. Beta Lactam Resistance .............................................................................................. 25 1.3.2.2. Aminoglycoside Resistance ........................................................................................ 30 1.3.2.3. Fluoroquinolone Resistance ........................................................................................ 30 1.3.3. Porins and Antimicrobial Resistance (AMR) ..................................................................... 31 1.3.4. Efflux and Antimicrobial Resistance .................................................................................. 33 1.4. The Role of Porins in Bacterial Pathogenesis ............................................................................. 35 1.5. C. elegans as a Model for Bacterial Pathogenesis ...................................................................... 40 1.5.1. C. elegans ............................................................................................................................ 40 1.5.2. Anatomy .............................................................................................................................. 42 ii 1.5.3. Immune System................................................................................................................... 44 1.5.4. Benefits of C. elegans as an Infection Model ..................................................................... 46 1.5.5. S. marcescens and C. elegans ............................................................................................. 47 1.6. Genomics of S. marcescens ........................................................................................................ 48 2. Outer Membrane Porins in S. marcescens ...................................................................................... 49 2.1. Materials and Methods ................................................................................................................ 49 2.1.1. Bacterial Strains and Plasmids ............................................................................................ 49 2.1.2. Media and Growth Conditions ............................................................................................ 54 2.1.3. Genomic DNA Isolation ..................................................................................................... 54 2.1.4. Preparation of Competent Cells .......................................................................................... 55 2.1.4.1. Chemical Competent Cell preparation using Rubidium chloride ............................... 55 2.1.4.2. Preparation of S. marcescens Db11 Electrocompetent Cells ...................................... 56 2.1.5. Restriction Digest and Cleanup of DNA products .............................................................. 56 2.1.6. Design of Unmarked Mutants ............................................................................................. 57 2.1.7. Preparation of total RNA .................................................................................................... 57 2.1.8. Quantitative Real-Time PCR (qPCR) ................................................................................. 59 2.1.9. Microscopy ......................................................................................................................... 63 2.1.10. Antibiotic Susceptibility Assay ........................................................................................... 63 2.1.11. C. elegans Survival Assays ................................................................................................. 64 2.1.11.1. Nematode Growth and Maintenance ........................................................................... 64 2.1.11.2. Preparation of S. marcescens assay plates .................................................................. 64 2.1.11.3. Survival assays in C. elegans ...................................................................................... 64 2.1.11.4. Survival Killing Assay with Galleria mellonella ........................................................ 65 2.2. Results ......................................................................................................................................... 66 2.2.1. S. marcescens Porin Mutant Growth Rate .......................................................................... 66 2.2.2. S. marcescens Porin Mutant Strain Virulence ..................................................................... 70 2.2.3. Visualization of C. elegans Colonization by S. marcescens Mutant Strains ...................... 72 2.2.4. S. marcescens Porin Mutant Strain Virulence in Galleria mellonella ................................ 78 2.2.5. Expression of OMP Genes in S. marcescens Mutant Strains .............................................. 80 2.2.6. Generating additional porin mutant strains ......................................................................... 81 3. S. marcescens Clinical Isolates ......................................................................................................... 83 3.1. Materials and Methods ................................................................................................................ 83 3.1.1. Preparation of Genomic DNA for Sequencing ................................................................... 83 iii 3.1.2. PacBio Next Generation Genome Sequencing ................................................................... 84 3.1.3. Assembly and Annotation ................................................................................................... 84 3.1.4. Comparison and Analysis ................................................................................................... 85 3.1.4.1. Phylogeny of Clinical Isolates .................................................................................... 85 3.1.4.2. Mobile Elements ........................................................................................................