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 Virulence Factors: Molecular Approaches for Host Factors 226

Comparative Approaches to Identify Host Factors Required for Infection 227

Transgenic Animal Models 228

In vivo Imaging of Animals during Infection 231

Systems Genetics: Comparative Genomics of the Host Response 231

Screening Approaches to Identify Host Factors Required for Infection 235

Genome-Wide Screening 235

Host Response Profiling to Identify Host Factors Required for Infection 240

Transcriptomics 240

Proteomics 243

Metabolomics 246

The Promise and the Caution 247

Selected Readings 248

Questions 249

Solving Problems in Bacterial Pathogenesis 249

Chapter 11 Bacterial Strategies for Colonization and Survival in the Host 254

What Does Not Kill You Makes You Stronger—Or, a Better Pathogen 255

Preinfection 258

Survival in the External Environment 258

Biofilms 258

Motility and Chemotaxis 260

Colonization of Host Surfaces 263

Penetrating Intact Skin 263

Penetrating the Mucin Layer 263

Evading the Host’s Innate Immunity 264

Nutrient and Iron Acquisition Mechanisms 265

Adherence 268

Evading the Host Immune Response 275

Avoiding Complement and Phagocytosis 277

Invasion and Uptake by Host Cells 280

Surviving Phagocytosis 281

Cell-to-Cell Spread 286

Tissue Penetration and Dissemination 288

Beyond Virulence Factors 288

Selected Readings 289

Questions 290

Solving Problems in Bacterial Pathogenesis 290

Special Global Perspective Problem: Integrating Concepts in Pathogenesis 291

Chapter 12 Toxins and Other Toxic Virulence Factors 294

Bacterial Toxins 295

Transparent Mechanisms, Exciting Applications, Mysterious Purposes 295

Toxin Characteristics and Nomenclature 300

Nonprotein Toxins 302

Peptide and Protein Exotoxins 304

Toxic Effector Proteins of Specialized Secretion Systems 312

Examples of Toxin-Mediated Diseases 313

Diphtheria Toxin 313

Clostridial Neurotoxins 319

Cholera Toxin 323

Toxin-Based Therapeutics and Research Tools 327

Immunotoxins 327

Selected Readings 329

Questions 330

Solving Problems in Bacterial Pathogenesis 331

Chapter 13 Delivery of Virulence Factors 334

Bacterial Secretion Systems and Virulence 335

Common Secretory Systems 336

The General Secretory (Sec) System 336

The Accessory Secretory (Sec) System 336

The Cotranslational Signal-Recognition Particle (SRP) System 336

The Twin-Arginine Transport (TAT) System 337

Secretion Systems Specific to Gram-Negative Bacteria 338

Sec-Dependent Secretion Systems 338

Sec-Independent Secretion Systems 341

Specialized Secretion Systems Specific to Gram-Positive Bacteria 350

General Secretory Transporter Systems in Gram-Positive Bacteria 350

Cytolysin-Mediated Translocation (CMT) in S. pyogenes (Group A Strep) 351

Type 7 Secretion System (T7SS) 352

Selected Readings 353

Questions 354

Solving Problems in Bacterial Pathogenesis 355

Chapter 14 Virulence Regulation 360

Virulence Gene Regulation 361

Mechanisms of Regulation 361

Operons, Regulons, and Global Regulators 362

Activators and Repressors 362

Two-Component Regulatory Systems 364

Sigma Factors 368

Transcriptional Terminators and Antiterminators 369

Regulation of Translation Initiation 372

Regulatory Small RNAs 372

Bacterial Chromatin 375

Responding to Environmental Signals 376

Phase Variation and Bistable Switches 376

Hypermutability, Intragenomic Recombination, and Positive Selection 377

Coordinate Virulence Regulation 377

Quorum Sensing 378

Chemotaxis 387

Selected Readings 389

Questions 389

Solving Problems in Bacterial Pathogenesis 390

Special Global Perspective Problems: Integrating Concepts in Pathogenesis 395

Chapter 15 Antimicrobial Compounds and Their Targets 400

Antimicrobial Compounds: The Safety Net of Modern Medicine 401

The Importance of Antimicrobial Compounds 401

Avoiding, Reducing, and Preventing Exposure 402

Killing versus Inhibiting Growth 404

Tests Used to Assess Antibiotics 404

Antiseptics and Disinfectants 405

Antibiotics 407

Characteristics of Antibiotics 407

The Process of Antibiotic Discovery 409

The Economics of Antibiotic Discovery 413

Mechanisms of Antibiotic Action 416

Targets of Antibiotic Action 416

Cell Wall Synthesis Inhibitors 417

Protein Synthesis Inhibitors 422

Antibiotics That Target DNA and RNA Synthesis 425

Inhibitors of Tetrahydrofolate Biosynthesis 429

The Newest Antibiotics 429

The Newest Antibiotic Targets 430

Strategies for Enhancing Antibiotic Efficacy 431

The Continuing Challenge 432

Selected Readings 433

Questions 434

Solving Problems in Bacterial Pathogenesis 435

Chapter 16 Antibiotic Resistance 436

The Dawning of Awareness—Uh, We Have a Problem! 437

How Did We Get to Where We Are? 438

And Now the Really Scary Part . . . 440

Emergence and Challenge of Multidrug Resistance (MDR) 440

Multiple Resistance and Genetic Linkage 441

Next-Generation MDR Pathogens: The “Superbugs”! 441

Mechanisms of Antibiotic Resistance 444

Overview of Resistance Mechanisms 444

Resistance to Antiseptics and Disinfectants 444

Limiting Access of the Antibiotic 445

Enzymatic Inactivation of the Antibiotic 446

Modification or Protection of the Antibiotic Target 451

Failure to Activate an Antibiotic 453

Regulation of Resistance Genes 454

Antibiotic Tolerance and Persister Cells 457

Antibiotic Tolerance 457

Persistence 459

Toxin-Antitoxin Systems 459

Horizontal Gene Transfer (HGT) of Resistance Genes 461

Propagating and Maintaining Antibiotic Resistance through Selective Pressure and Changes in Fitness 463

Will We Return to the Pre-Antibiotic Era? 464

Returning to Status Quo or Moving Forward? 464

The Hunt for Alternative Approaches to Antibiotics 466

Selected Readings 468

Questions 469

Solving Problems in Bacterial Pathogenesis 470

Special Global Perspective Problems: Integrating Concepts in Pathogenesis 470

Chapter 17 Vaccination: A Critical Component of the Modern Medical Armamentarium 476

Vaccines: A Major Health Care Bargain 477

What Makes an Ideal Vaccine? 479

Immunization Programs 480

Barriers to Implementation and Success of Immunization Programs 483

The Antivaccination Movement 484

Vaccine Success Stories 485

Subunit Vaccines 485

Conjugate Vaccines 490

Vaccine “Less-than-Success” Stories 492

A New Age of Vaccine Development: Making Vaccines Better 494

Approaches to Enhancing Immunogenicity 494

Adjuvants 494

Programming Adaptive Immunity 496

Targeting Mucosal Immunity 500

Storage of Vaccines—Strategies to Increase Shelf Life 504

Passive Immunization 504

Selected Readings 505

Questions 505

Solving Problems in Bacterial Pathogenesis 508

Special Global Perspective Problems: Integrating Concepts in Pathogenesis 510

Chapter 18 The Gram-Positive Opportunistic Pathogens 514

What Is an Opportunist? 515

Characteristics of Gram-Positive Opportunists 516

Notable Gram-Positive Opportunists 516

Staphylococcus aureus—Commensal Ready for a Fight 516

Staphylococcus epidermidis—Accidental Pathogen 524

Streptococcus pneumoniae—“Captain of All the Men of Death” 527

Clostridium difficile—True Opportunist 534

Other Gram-Positive Opportunists 542

Selected Readings 543

Questions 544

Solving Problems in Bacterial Pathogenesis 544

Chapter 19 The Gram-Negative Opportunistic Pathogens 548

Jumping Over the (Cell) Wall: Gram-Negative Bacteria Can Be Opportunistic Pathogens Too! 549

Common Traits of Gram-Negative Opportunists 549

The Dark Side of Some Residents of the Human Body 550

The Ever-Changing Face of E. coli 550

Klebsiella pneumoniae Nosocomial Infections 554

Bacteroides fragilis—The Bad Sheep of the Family 555

Porphyromonas gingivalis—A Keystone Pathogen 557

Environmental Inhabitants Weigh in as Opportunists 558

Pseudomonas aeruginosa—A Versatile Opportunist of the Highest Order 558

Burkholderia cepacia Complex—P. aeruginosa’s Evil Twin 565

Acinetobacter baumannii—A Deadly Threat Emerges from the Iraq War 565

Don’t Forget the Arthropods! 567

Ehrlichia spp. 567

Selected Readings 568

Questions 569

Solving Problems in Bacterial Pathogenesis 569

Special Global Perspective Problems: Integrating Concepts in Pathogenesis 570

Chapter 20 The Changing Roles of Microbiologists in an Age of Bioterrorism and Emerging Diseases 574

When Microbiologists Are Called to the Front Line 575

Tracking Down a Bioterrorist 576

Unintentional or Deliberate? 577

Timing 579

Tracing the Source 579

Lessons Learned 579

The “Top Four” Bioterror Agents 582

Bacillus anthracis Spores 582

Smallpox 583

Yersinia pestis 583

Botulinum Neurotoxin 584

What If Bioterrorists Hit Us with Something Completely New? 585

Biosecurity in a Complex, Dynamic, and Ever-Changing World 585

Food Safety and Biosecurity 586

The Case for Food Irradiation 587

The Future of Biosecurity 589

Selected Readings 589

Questions 590

Solving Problems in Bacterial Pathogenesis 591

Glossary 593

Index 661