Phylogeny,Classification antimicrobial, antimicrobial susceptibility susceptibility and and virulence factors of Aeromonas species in virulence factors of Western Australian Western Australia By Max Aravena-Román BScAppSci FASM This thesis is presented for the degree of Doctor of Philosophy School of Pathology and Laboratory Medicine of Western Australia 2015 TABLE OF CONTENTS TABLE OF CONTENTS.................................................................................................i SUMMARY.....................................................................................................................ix DECLARATION............................................................................................................xi ACKNOWLEDGEMENTS.........................................................................................xiii THESIS STRUCTURE.................................................................................................xv ABBREVIATIONS.......................................................................................................xvi LIST OF TABLETS......................................................................................................xx LIST OF FIGURES...................................................................................................xxiii CHAPTER 1: LITERATURE REVIEW..........................................................1 1.1. GENERAL INTRODUCTION.......................................................................1 1.2. HISTORY........................................................................................................1 1.3. TAXONOMY..................................................................................................2 1.3.1. Early taxonomy..........................................................................................2 1.3.2. Current taxonomy......................................................................................3 1.3.3. Controversial taxonomic issues.................................................................3 1.3.3.1. Aeromonas allosaccharophila..............................................................4 1.3.3.2. Aeromonas spp. HG 11........................................................................8 1.3.3.3. Aeromonas culicicola...........................................................................8 1.4. LABORATORY IDENTIFICATION...........................................................9 1.4.1. Isolation.....................................................................................................9 1.4.2. Identification by phenotypic methods.....................................................12 1.4.3. Identification by commercial systems.....................................................13 1.4.4. Additional phenotypic methods..............................................................15 1.4.5. Semi-automated systems.........................................................................15 1.4.6. Identification by molecular methods.......................................................16 1.4.6.1. Typing methods………………………………………......................16 1.4.6.2. Identificartion based on 16S-23S rRNA gene sequence……………17 1.4.6.3. Identification based on housekeeping gene sequence........................18 1.4.6.4. Specific genes used as identification targets......................................18 1.4.6.5. Restriction enzyme-based methods....................................................19 1.4.6.6. PCR-based methods............................................................................20 1.4.6.7. Disadvantages of molecular methods.................................................20 1.5. SEROTYPING............................................................................................21 1.6. ECOLOGY..................................................................................................22 -i- 1.6.1. Aquatic environments.............................................................................22 1.6.1.1. Distribution in water...............................................................................22 1.6.1.2. Water quality...........................................................................................23 1.6.1.3. Effects of temperature on growth and toxin production.........................24 1.6.1.4. Aeromonas in drinking water..................................................................25 1.6.2. Aeromonas in foods.................................................................................28 1.6.2.1 Distribution of Aeromonas spp. in foods............................................28 1.7. EPIDEMIOLOGY AND PUBLIC HEALTH ISSUES............................30 1.7.1. Water-associated infections.....................................................................31 1.7.2. Foods-associated infections.....................................................................32 1.7.3. Aeromonas and fish infections................................................................32 1.8. BIOREMEDIAL AND BIODEGRADABLE PROPERTIES....................33 1.9. VIRULENCE FACTORS...........................................................................34 1.9.1. Adherence................................................................................................35 1.9.2. Pili............................................................................................................36 1.9.3. Invasins....................................................................................................39 1.9.4. S-layer......................................................................................................40 1.9.4.1. Structural arrangements......................................................................41 1.9.4.2. Binding properties..............................................................................41 1.9.4.3. Genes involved in S-layer synthesis...................................................41 1.9.4.4. S-layer and virulence..........................................................................42 1.9.5. The lipopolysaccharide (LPS).................................................................42 1.9.5.1. Functions of the LPS.........................................................................42 1.9.5.2. Immunological and antigenic properties of the LPS.........................43 1.9.5.3. Genes involved in LPS synthesis.......................................................43 1.9.6. Outer membrane proteins (OMP)............................................................44 1.9.7. Flagella....................................................................................................45 1.9.7.1. Synthesis, regulation and expression of flagella................................45 1.9.7.2. Functions associated with flagella......................................................46 1.9.8. Secretion systems....................................................................................46 1.9.8.1. Type II secretion systems (T2SS).......................................................48 1.9.8.2. Type III secretion systems (T3SS).....................................................48 1.9.8.3. Type IV secretion systems (T4SS).....................................................49 1.9.8.4. Type VI secretion systems (T6SS).....................................................50 1.9.9. Exotoxins................................................................................................51 -ii- 1.9.9.1. Aerolysin...........................................................................................52 1.9.9.1.1. Action on host tissue..................................................................53 1.9.9.1.2. Molecular characteristics and prevalence...................................53 1.9.9.2. Cytotoxic enterotoxin (Act)...............................................................54 1.9.9.3. Haemolysins.......................................................................................55 1.9.9.4. Enterotoxins.......................................................................................56 1.10. Additional extracellular products............................................................59 1.10.1. Proteases.............................................................................................59 1.10.2. Lipases................................................................................................60 1.10.3. Nucleases (DNases)............................................................................61 1.10.4. Chitinases............................................................................................62 1.11. Iron uptake...............................................................................................63 1.12. Quorum sensing (QS)..............................................................................64 1.13. Biofilm formation....................................................................................65 1.14. Additional virulence factors....................................................................66 1.15. INFECTIONS CAUSED BY AEROMONAS SPECIES........................67 1.15.1. Gastroenteritis.....................................................................................68 1.15.1.1. Disease presentation....................................................................68 1.15.1.2. Evidence against Aeromonas as an enteric pathogen..................69 1.15.1.3. Evidence supporting Aeromonas as an enteric pathogen............71 1.15.1.4. Species