Brenda A. Wilson, Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana-Champaign, Illinois.

Malcolm E. Winkler, Department of Biology, Indiana University, Bloomington, Indiana.

Brian T. Ho, Institute of Structural and Molecular Biology, University College London and Birkbeck, University of London, London, United Kingdom.

Preface xvii

In Memoriam xix

About the Authors xxi

Chapter 1 The Power of Bacteria 2

Why Are Bacteria So Much in the Public Health Spotlight Nowadays? 3

Bacteria, a Formidable Ancient Life Form 4

Pressing Current Infectious Disease Issues 6

Emerging and Reemerging Infectious Diseases 6

Foodborne and Waterborne Infections 7

Modern Medicine as a Source of New Diseases 8

Postsurgical and Other Wound Infections 9

Bioterrorism 10

A New Respect for Prevention 10

Surveillance: An Early Warning System 11

Making Hospitals Safe for Patients 12

And Now for Some Good News: You've Got a Bacterial Infection! 12

The Helicobacter pylori Revolution 12

The Aftermath 13

Microbiota Shift Diseases 13

A Brave New World of Pathogenesis Research 14

The New Age of Genomics 16

Insights into Pathogen Evolution 17

Modeling the Host-Pathogen Interaction in Experimental Animals 17

Correlation Studies 18

Selected Readings 18

Questions 20

Chapter 2 Skin and Mucosa: The First Lines of Defense against Bacterial Infections 22

The Best Defense: Avoid, Reduce, and Prevent Exposure! 23

Barriers: Skin and Mucosal Membranes 24

The Layers of Cells That Protect the Body 24

Normal Microbiota of the Skin and Mucosa 27

Defenses of the Skin 31

Defenses of Mucosal Surfaces 32

Special Defenses of the Gastrointestinal Tract 34

Special Defenses of the Urogenital Tract 36

Special Defenses of the Respiratory Tract 36

Immune Defenses of the Skin and Mucosa 37

Models for Studying Breaches of Barrier Defenses 38

Selected Readings 39

Questions 39

Chapter 3 The Innate Immune System: Always on Guard 40

Triggering Innate Immune Defenses 41

Innate Immune Cells That Defend Blood and Tissue 42

Neutrophils (PMNs) 42

Monocytes, Macrophages, and Dendritic Cells (DCs) 42

Granulocytes: Basophils, Mast Cells, and Eosinophils 47

Transmigration-How Do Phagocytes Know When and Where to Go? 47

Natural Killer (NK) Cells 48

The Lymphatic System 50

How Phagocytes Recognize and Respond to Bacteria 50

How Phagocytes Kill Bacteria 54

Oxidative Burst in Phagolysosomes 54

Autophagy-Another Pathway for the Killing of Intracellular Pathogens 56

The Complement Cascade 57

Complement Proteins 57

Overview of Complement Pathways and Their Function 58

Steps in Complement Activation 60

Controlling Complement Activation 62

Cytokines and Chemokines-Mediators of Immune Responses 63

Roles of Cytokines and Chemokines in Directing Innate Immune Responses 63

Inflammation and Collateral Damage 65

Septic Shock: The Dark Side of the Innate Defenses 66

Other Innate Defenses of the Body-Nutritional Immunity 69

Selected Readings 70

Questions 70

Chapter 4 The Adaptive Defenses: Antibodies and Cytotoxic T Cells 72

The Specialists: Adapting to a Particular Pathogen Challenge 73

B Cells: Producers of Antibodies 74

The Humoral (Antibody) Immune Response 74

Characteristics of Antibodies and Their Diverse Roles in Preventing Infection 74

Serum Antibodies 76

Secretory Antibodies: Antibodies That Protect Mucosal Surfaces 78

Pathogen and Toxin Neutralization by Antibodies 79

Affinity and Avidity 80

Cytotoxic T Cells, Also Known as Cytotoxic T Lymphocytes (CTLs) 81

Cytotoxic T Lymphocytes: Critical Defense against Intracellular Pathogens 81

Antigen Presentation to the Immune System 82

Processing of Protein Antigens by Dendritic Cells 82

Interaction between APCs and T Cells: The T-Cell-Dependent Response 84

Th-(Th1/Th2/Th17)-Cell-Mediated Immunity 86

Production of Antibodies by B Cells 87

Links between the Innate and Adaptive Defense Systems 88

T-Cell-Independent Antibody Responses 89

Mucosal Immunity: IgA/sIgA Antibodies 89

Development of the Adaptive Immune System from Infancy to Adulthood 92

Adaptive Defense Systems in Nonmammals 93

The Dark Side of the Adaptive Defenses: Autoimmune Disease 93

Selected Readings 94

Questions 94

Solving Problems in Bacterial Pathogenesis 95

Chapter 5 The Microbiota of the Human Body: Microbiomes and Beyond 98

Importance of the Normal Resident Microbial Populations (Microbiota) of the Human Body 99

Characterization of the Body's Microbiota 100

Taking a Microbial Census by Using Microbial rRNA Gene Sequence Analysis 101

Characterizing Microbiomes by Using Metagenomic Analysis 115

Beyond the Metagenome 117

Overview of the Human Microbiota 123

Skin Microbiota 124

Oropharyngeal Microbiota 125

Microbiota of the Small Intestine and Colon 125

Microbiota of the Vaginal Tract 128

The Other Microbiota: The Forgotten Eukaryotes 130

Selected Readings 130

Questions 131

Solving Problems in Bacterial Pathogenesis 133

Chapter 6 Microbes and Disease: Establishing a Connection 134

History and Relevance of Koch's Postulates 136

Early Germ Theory 136

Koch's Postulates: A Set of Criteria Used to Establish a Microbe-Disease Connection 137

Challenges to Satisfying Koch's Postulates 138

Easier Said than Done . . .¿ 138

The First Postulate: Association of the Microbe with Lesions of the Disease 139

The Second Postulate: Isolating the Bacterium in Pure Culture 140

The Third Postulate: Showing that the Isolated Bacterium Causes Disease Experimentally in Humans or Animals 141

The Fourth Postulate: Reisolating the Bacterium from the Intentionally Infected Animal 143

Modern Alternatives to Satisfy Koch's Postulates 143

Detecting the Presence of the Pathogen Only in Diseased Tissues 143

Eliminate the Pathogen and Prevent or Cure the Disease 144

Comparative Infectious Disease Causation 147

The Microbiota Shift Disease Problem 147

Koch's Postulates and Pathogenic Microbial Communities 147

Keystone Pathogens and Microbial Shift Diseases 147

Molecular Koch's Postulates 149

Concepts of Disease 150

Varieties of Human-Microbe Interactions 150

Views of the Human-Microbe Interaction 150

Virulence as a Complex Phenomenon 152

Selected Readings 152

Questions 153

Solving Problems in Bacterial Pathogenesis 153

Chapter 7 Mechanisms of Genetic Modification and Exchange: Role in Pathogen Evolution 156

Adapt or Perish 158

Acquiring New Virulence Traits by Horizontal Gene Transfer 158

Mechanisms of Genetic Change and Diversification 158

Spontaneous Mutation 158

Phase Variation 159

Antigenic Variation 161

Horizontal Gene Transfer: Mobile Genetic Elements 162

Natural Transformation 162

Conjugation: Plasmids and Transposons 165

Phage Transduction 171

Control of Horizontal Gene Transfer 171

Toxin-Antitoxin Systems-Retaining the Goods 171

Restriction-Modification Systems-Bacterial Innate Immunity from Foreign DNA 173

CRISPR-Cas Systems-Bacterial Adaptive Immunity from Foreign DNA 173

Type 6 Secretion Systems-Bacterial Defense Against Conjugation 174

Pathogenicity Islands and Pathogen Evolution 174

Properties of Pathogenicity Islands 174

Pathogen Evolution in Quantum Leaps 177

Selected Readings 178

Questions 179

Solving Problems in Bacterial Pathogenesis 179

Chapter 8 Identification of Virulence Factors: Measuring Infectivity and Virulence 182

How Does One Experimentally Measure Virulence and Satisfy Koch's Postulates? 183

Animal Models of Infection 184

Human Volunteers 184

Nonhuman Animal Models 186

Measuring Bacterial Infection in Animal Models 188

Ethical Considerations 188

Animal Model Basics 188

Survival Curve Analysis and Biophotonic Imaging 189

ID50 and LD50 Values 190

Competition Assays 191

Tissue Culture and Organ Culture Models 192

Tissue Culture Models 192

Gentamicin Protection Assay for Cell Adhesion and Invasion 193

Plaque Assay for Intracellular Survival and Cell-to-Cell Spread 195

Fluorescence Microscopy Techniques for Assessing Effects of Pathogens on Host Cells 196

Organ Culture Models 196

The Continuing Need for Reliable and Plentiful Information about Disease Pathology 197

Selected Readings 198

Questions 199

Solving Problems in Bacterial Pathogenesis 200

Chapter 9 Identification of Virulence Factors: Molecular Approaches for Bacterial Factors 202

Finding a Needle in a Haystack 204

Biochemical Approaches 204

Isolation and Purification of Toxic Factors 204

Molecular Genetic Approaches 208

Screening Using Recombinant Genes 208

Reporter Fusions 208

Mutagenesis Screening 210

Genome-wide Sequencing Approaches for Identifying Virulence Genes 214

Tn-Seq Technology to Identify In Vivo-Expressed Genes 214

RNA-Seq Technology to Identify In Vivo-Expressed Genes 216

Comparative Genomic Sequence Analysis for Identifying Virulence Genes 217

Proteomics Approaches for Identifying Virulence Factors 218

Protein Microarrays (Proteoarrays) 218

In Vivo-Induced Antigen Technology (IVIAT) 218

The Importance of Understanding Bacterial Physiology 219

Selected Readings 222

Questions 223

Solving Problems in Bacterial Pathogenesis 223

Chapter 10 Identification of...