Preface
Contents
Contributors
Part I: An Overview of the Diversity of Bacteriophages
Chapter 1: Structural and Genomic Diversity of Bacteriophages
1 Introduction
2 Bacteriophage Taxonomy
3 dsDNA Phages
4 ssDNA Phages
5 RNA Phages
6 Phage Discovery
7 Bacteriophage Host Range
8 Phage Genome Comparisons
9 Phage Genome Structure
10 Conclusions
References
Chapter 2: The Diversity of Bacteriophages in the Human Gut
1 Introduction
2 Methods for Detection and Identification of Phages
3 Diversity of Phages in the GIT
3.1 Compositional Diversity
3.2 Temporal Diversity
3.3 Spatial Diversity
3.4 Functional Diversity
4 Future Perspectives
5 Conclusions
References
Chapter 3: Breaking the Ice: A Review of Phages in Polar Ecosystems
1 Introduction
2 A Closer Look at the Arctic and Antarctica as Viral Territory
2.1 Characteristics of Polar Environments
2.2 A General Introduction to Viruses in Polar Ecosystems
3 Challenges Related to Sampling and Analyzing Phages from Polar Regions
4 Insights from -Omics Approaches to Identify Viruses
4.1 The Benefits of -Omics Approaches in Polar Virology
4.2 AMGs and Molecular Adaptations of Viruses in Cold Environments
5 Cultivation of Phage from the Cryosphere
5.1 Synopsis of the Merits and Successes of Phage Cultivation
5.2 Cold-Adapted Phages and How to Cultivate Them
6 Viral Abundance and Diversity of Different Polar Ecosystems
6.1 Viruses from Marine Ecosystems
6.2 Viruses in Sea Ice
6.3 Viruses in the Polar Atmosphere
6.4 Viral Abundance and Diversity in Polar Freshwater Environments
6.4.1 Viruses in Polar Lakes
6.4.2 Viruses in Cryoconite Holes
6.5 Viruses from Soil and Peatlands
7 Ecology of Polar Phages
7.1 Strategies, Interactions, and Impact of Polar Phages in the Environment
7.1.1 Predominant Replication Modes
7.1.2 Growth and Survival of Polar Phages in the Environment
7.1.3 Ecological Consequences of Phage Infections for Microbial Evolution, Community Dynamics, and Biogeochemical Cycles
7.2 Dispersal of Viruses to and from the Poles
8 Major Challenges, Knowledge Gaps, and Future Perspectives in Polar Phage Research
8.1 Major Challenges in Polar Phage Research
8.2 Major Knowledge Gaps in Polar Phage Research
8.3 Future Perspectives
References
Chapter 4: The Diversity of Bacteriophages in Hot Springs
1 Ecology of Hot Springs
1.1 Environmental Conditions
1.2 Microbial Population
1.3 Adaptations of Thermophiles for Survival
2 Bacteriophages of Hot Springs
2.1 Overview
2.2 Myoviridae
2.3 Siphoviridae
2.4 Tectiviridae
2.5 Sphaerolipoviridae
2.6 Inoviridae
3 Uncultivated
4 Biotechnological Importance of Thermophilic Bacteriophages
4.1 Industrial Potential of Thermophilic Bacteriophages
4.2 Recombineering
4.3 Medical Potential of Thermophilic Bacteriophages
References
Part II: Isolation of Bacteriophages
Chapter 5: Isolation of Bacteriophages from Soil Samples in a Poorly Equipped Field Laboratory in Kruger National Park
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffer and Media Preparations
3 Methods
3.1 Sample Preparation
3.2 Isolation of Soil Bacteriophages
3.3 Phage Propagation and Plaque Identification
3.4 Harvesting Bacteriophage from Plaques
3.5 Plaque Enumeration/Titer Determination
3.6 Host Specificity Determination
3.7 Bacteriophage Induction from Soil Bacteria
3.8 Microscopy to Evaluate the Killing of Host Bacteria by Isolated Phage
3.9 Phage Purification (by PEG Precipitation) for Transmission Electron Microscopy
4 Notes
References
Chapter 6: Purification and Up-Concentration of Bacteriophages and Viruses from Fecal Samples
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
3 Methods
4 Notes
References
Chapter 7: Isolation of Enterococcus Bacteriophages from Municipal Wastewater Samples Using an Enrichment Step
1 Introduction
2 Materials
2.1 Collection of Samples
2.2 Solutions and Buffers
2.3 Equipment and Materials
3 Methods
3.1 Sample Collection
3.2 Sample Clarification and Fractionation
3.3 Enrichment and Host Isolation
3.4 PEG Precipitation (Optional)
3.5 Phage Isolation
4 Notes
References
Chapter 8: Phage DNA Extraction, Genome Assembly, and Genome Closure
1 Introduction
1.1 Phage Lysate Preparation and Phage DNA Extraction
1.2 Phage Genome Sequencing and Assembly
1.3 Phage Genome Closure from the Assembled Contig
2 Materials
2.1 Phage Lysate Preparation and Phage DNA Extraction
2.2 Phage Genome Sequencing and Assembly
2.3 Standard PCR
3 Methods
3.1 Preparation of High-Titer Phage Lysate for DNA Extraction
3.2 Wizard DNA Cleanup Kit-Based DNA Extraction
3.3 Phenol-Chloroform DNA Extraction
3.4 Data Upload and Assessment in FastQC
3.5 Read Quality Control with Trimmomatic
3.6 Read Assembly Using SPAdes
3.7 Closure PCR to Amplify the Gap Region Joining the Contig Ends
3.8 Direct Sequencing of Phage DNA Ends
4 Notes
References
Part III: Enumeration of Bacteriophages
Chapter 9: Enumeration of Bacteriophages by Plaque Assay
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Bacteria and Bacteriophage
3.2 Dilutions of Bacteriophage
3.3 Plating of Bacteriophage
3.4 Observation
4 Notes
References
Chapter 10: Detection and Quantification of Bacteriophages in Wastewater Samples by Culture and Molecular Methods
1 Introduction
2 Materials
2.1 Isolation and Quantification of Reference Bacteriophages in Wastewater
2.1.1 Host Strain Stock Culture Preparation
2.1.2 Host Strain Inoculum Culture Preparation for Bacteriophage Quantification
2.1.3 Enumeration of Reference Bacteriophages by a Double Agar Layer Plaque Assay
2.1.4 Bacteriophage Isolation from Plaques
2.1.5 High-Titer Bacteriophage Suspension Preparation
2.1.6 Reference Bacteriophage Stock Preparation
2.2 Isolation and Quantification of Somatic Coliphages
2.3 Isolation and Quantification of F-Specific Bacteriophages
2.4 Isolation and Quantification of Bacteroides (Anaerobic Bacteria) Bacteriophages
2.5 Extraction of DNA from Bacteriophages
2.6 Absolute Quantification of Bacteriophages by Molecular Methods
3 Methods
3.1 Isolation and Quantification of Reference Bacteriophages in Wastewater
3.1.1 Host Strain Stock Culture Preparation
3.1.2 Host Strain Inoculum Culture Preparation for Bacteriophage Quantification
3.1.3 Enumeration of Target Bacteriophages by a Double Agar Layer Plaque Assay (Fig. 1)
3.1.4 Bacteriophage Isolation from Plaques
3.1.5 High-Titer Bacteriophage Suspension Preparation
3.1.6 Reference Bacteriophage Stock Preparation
3.2 Extraction of DNA from Bacteriophages
3.3 Absolute Quantification of Bacteriophages by Molecular Methods
4 Notes
References
Chapter 11: Flow Virometry: A Fluorescence-Based Approach to Enumerate Bacteriophages in Liquid Samples
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Microbiological Media, Bacteriophages, and Host Bacteria
2.3 Other Materials
2.4 Equipment
3 Methods
3.1 Preparation of Standard Bacteriophage and Host-Control Cultures
3.2 Preparing Host-Control and Bacteriophage Samples for Virometry
3.3 Preparing Water Samples for Virometry
3.4 Virometry
4 Notes
References
Chapter 12: A Metagenomics Approach to Enumerate Bacteriophages in a Food Niche
1 Introduction
2 Materials
2.1 Materials and Equipment
2.2 Preparation of Solutions
2.3 Whey Samples
3 Methods
3.1 Sample Preparation (Adapted from)
3.2 DNA Isolation
3.3 Preparation of Viral DNA Sample for Sequencing: Library Construction
3.3.1 Fragmentation of DNA Sample
3.3.2 Amplification of Libraries
3.3.3 Clean-up of Libraries
3.3.4 Library Quality Check and Normalization of Libraries
3.4 DNA Sequencing
3.5 In Silico DNA Sequence Analysis
3.6 Phageome Taxonomic Analysis through Reads Mapping
3.6.1 Create Phage Database(S)
3.6.2 Filter Raw Reads
3.6.3 Reads Mapping and Subsequent Calculation
4 Notes
References
Part IV: Characterization of Bacteriophages
Chapter 13: Bioinformatic Analysis of Staphylococcus Phages: A Key Step for Safe Cocktail Development
1 Introduction
2 Materials
3 Methods
3.1 Genome Annotation
3.2 Construction of OLD (Our Local Database)
3.3 Phylogenetic Analysis
4 Notes
References
Chapter 14: Use of Localized Reconstruction to Visualize the Shigella Phage Sf6 Tail Apparatus
1 Introduction
2 Materials
2.1 Vitrification
2.2 Cryogenic Electron Microscopy
2.3 Single-Particle Analysis
2.4 Model Building and Analysis Software
3 Methods
3.1 Vitrification
3.2 Cryogenic Electron Microscopy
3.3 Single-Particle Analysis
3.4 Model-Building
4 Notes
References
Chapter 15: Bacteriophage-Host Interactions and Coevolution
1 Introduction
2 First Phage-Bacteria Interaction
3 Resistance to Phage Action and Counter-Resistance
4 Ecological Models to Study Phage-Bacteria Interactions
5 Evolutionary Models for Phage-Bacteria Interactions in the Environment
6 Current Models of Bacteriophage Coevolution in the Gut
7 Conclusion
References
Chapter 16: Unraveling Physical Interactions of Clostridioides difficile with Phage and Phage-Derived Proteins Using In Vitro ...
1 Introduction
2 Materials and Reagents
2.1 Clostridioides difficile Culture
2.2 Preparation of Cell Wall Polymers
2.3 Expression and Purification of Recombinant Proteins
2.4 Pull Down Using Peptidoglycan-Polysaccharide (PG-PS) Complex
2.5 Far Immunoblotting Assay
2.6 Determining the Bacterial Cell Binding Using Fluorescent Fusion Protein
2.7 Determining Bacterial Cell Binding Using Whole-Cell Enzyme-Linked Immunosorbent Assay (ELISA)
3 Methods
3.1 Clostridioides difficile Culture
3.2 Preparation of Cell Wall Polymer
3.2.1 Extraction of Peptidoglycan-Polysaccharide (PG-PS) Complex
3.2.2 Preparation of Peptidoglycan (PG)
3.2.3 Polysaccharide (PS) Extraction
3.2.4 PS Quantification
3.3 Protein Expression and Purification
3.3.1 Bacterial Culture and Induction
3.3.2 Bacterial Cell Lysis
3.3.3 Column Equilibration
3.3.4 Protein Loading and Purification
3.3.5 Column Storage
3.4 Pull Down Using PG-PS
3.5 Far Immunoblotting Assay
3.6 Determining Bacterial Cell Binding Using Fluorescent Protein
3.7 Determining Bacterial Cell Binding Using Whole-Cell Enzyme-Linked Immunosorbent Assay (ELISA)
4 Notes
References
Chapter 17: Phage Transduction of Staphylococcus aureus
1 Introduction
2 Materials
2.1 S. aureus Strains and Transducing Phages
2.2 For Phage Lysates
2.3 For Phage Titers
2.4 For Transducing Particle Lysate
2.5 For Phage Transduction Assay
2.6 For Confirmation of Transductants and Testing for Lysogeny
2.7 List of Consumables and Equipment
3 Methods
3.1 Generating a Phage Lysate from a Lysogenic S. aureus Strain
3.2 Determining Phage Titer
3.3 Propagating a Phage Lysate by Infection
3.4 Preparation of a Transducing Particle Lysate
3.5 Phage Transduction
3.6 Confirmation of Transductants by PCR Analysis
3.7 Testing for Lysogeny in Transductants
3.7.1 Testing for Lysogeny by Phage Immunity
3.7.2 PCR Analysis for Lysogeny
4 Notes
References
Part V: Application of Bacteriophages and Bacteriophage-Derived Components
Chapter 18: The Next Generation of Drug Delivery: Harnessing the Power of Bacteriophages
1 Introduction
2 Considerations in Manufacturing Bacteriophage-Loaded Biomaterials
3 Importance of Phages in Biotechnology
4 A Brief Overview of the Use of Bacteriophages for Cargo Delivery to Cells
4.1 Bacteriophages as a Delivery System
4.2 Advantages of Using Bacteriophages
4.2.1 Target Specificity
4.2.2 High Efficiency
4.2.3 No Immunological Response
5 Methods of Cargo Delivery Using Bacteriophages
5.1 Phage Display Technology
5.2 Encapsulation of the Cargo in Bacteriophage Particles
6 Applications of Bacteriophage-Mediated Delivery
6.1 Delivery of Antisense Oligonucleotides
6.2 Proteins and Peptides
6.3 Antibiotics
6.4 Delivery of Diagnostic Agents
6.5 Biosensors
7 Challenges and Limitations
7.1 Host specificity of Bacteriophages
7.2 Safety Concerns
7.3 Technical Limitations
7.4 Discussion of Future Directions and Potential Applications
8 Importance of Further Research in This Field
9 Conclusion
References
Chapter 19: Construction of Nonnatural Cysteine-Cross-Linked Phage Libraries
1 Introduction
2 Materials
2.1 Media, Solutions, and Bacterial Colonies
2.2 Equipment, Consumables, and Kits
2.3 M13KE Vector
2.4 Preparation of TOP10 Electrocompetent Cells and Testing of Electrocompetence
2.5 Oligonucleotide Design
2.6 Eight Percent Nondenaturing Polyacrylamide Gel
2.7 One Percent Agarose Gel
2.8 Phage Modification and ELISA Reagents
3 Methods
3.1 Preparing Oligonucleotide Duplex
3.2 Preparing M13KE Vector
3.3 Ligation and Transformation of M13KE Vector
3.4 Chemical Modification on Phage: 1,3-Dichloroacetone (DCA)
3.5 Modification Validation: Pulse-Chase ELISA
4 Notes
References
Chapter 20: Application of Deep Sequencing in Phage Display
1 Introduction
2 Materials
2.1 Phage Display
2.2 Sequencing
2.3 Sequence Analysis
3 Methods
3.1 Phage Display (Protocols Adapted from Yu & Smith, 1996)
3.2 Illumina Sequencing of Phagemid DNA
3.3 Oxford Nanopore Sequencing of Phagemid Libraries
4 Notes
References
Chapter 21: The Application of Bacteriophage and Photoacoustic Flow Cytometry in Bacterial Identification
1 Introduction
2 Materials
2.1 Blood Separation
2.2 Bacterial Culturing for Spiked Cultures
2.3 Bacteriophage
2.4 Photoacoustic Flow Cytometry
3 Methods
3.1 Blood Separation
3.2 Bacterial Culturing
3.3 Bacteriophage
3.4 Photoacoustic Flow Cytometry
4 Notes
References
22: Propagation, Purification, and Characterization of Bacteriophages for Phage Therapy
1 Introduction
2 Materials
2.1 Phage Titration
2.2 Bacteriophage Lysate Preparation in Liquid Culture
2.3 Phage Propagation on Solid Media
2.4 Polyethylene Glycol (PEG) Precipitation
2.5 Bacteriophage Purification by Cesium Chloride (CsCl) Density Gradient Ultracentrifugation
2.6 Bacteriophage Purification by Cesium Sucrose Density Gradient Ultracentrifugation
2.7 The Organic Solvent Purification (1-Octanol or 1-Butanol)
2.8 Basic Microbiological Characterizations
2.9 Adsorption Rate
2.10 One-Step Growth
2.11 Streak Tests
2.12 Lysogenization, Superinfection Resistance, and Prophage Induction
2.13 General Transduction Using Bacteriophage P1vir
2.14 M13 Lysate Preparation
2.15 Bacteriophage Isolation from Environmental Samples
2.15.1 Isolation of Bacteriophages from Sewage or Water Samples
2.15.2 Isolation of Phage from Soil Samples
2.15.3 Collection of Phage from Human Fecal Samples
2.16 Estimation of Phage Antibiofilm Activity
2.17 Assessment of Phage Therapeutic Potential Using Galleria mellonella Model
3 Methods
3.1 Phage Titration
3.1.1 Full-Plate Titration
3.1.2 Spot Test
3.2 Bacteriophage Lysate Preparation in Liquid Media
3.3 Phage Propagation on Solid Media
3.4 Polyethylene Glycol (PEG) Precipitation
3.5 Bacteriophage Purification by Cesium Chloride (CsCl) Density Gradient Ultracentrifugation
3.6 Bacteriophage Purification by Sucrose Density Gradient Ultracentrifugation
3.7 Organic Solvent Purification Using 1-Octanol or 1-Butanol
3.8 Basic Microbiological Characterizations
3.8.1 Lysis Profile
3.8.2 Colony-Forming Unit (CFU) and Plaque-Forming Unit (PFU) Assays
3.9 Adsorption Rate
3.10 One-Step Growth Experiment
3.11 Streak Tests
3.11.1 Streak Plate
3.11.2 Spots on Streaks
3.11.3 Cross-Streaking
3.12 Lysogenization, Superinfection Resistance, and Prophage Induction
3.12.1 Lysogenization
3.12.2 Superinfection Resistance Assay
3.12.3 Prophage Induction
3.13 Generalized Transduction Using P1vir
3.14 M13 Lysate Preparation
3.15 Bacteriophage Isolation from Environmental Samples
3.15.1 Isolation of Bacteriophages from Sewage or Water Samples
3.15.2 Isolation of Phages from Soil Samples
3.15.3 Isolation of Phages from Human Fecal Samples
3.16 Estimation of Phage Antibiofilm Activity
3.17 Assessment of Phage Therapeutic Potential Using Galleria mellonella Model
4 Notes
References
Chapter 23: Overcoming Bacteriophage Resistance in Phage Therapy
1 Introduction
2 How Does Phage Resistance Emerge?
3 Possible Ways to Overcome Phage Resistance
3.1 Administration of Multiple Phages Simultaneously or Sequentially
3.2 Synergistic Effect of Antibiotics and Phages
3.3 Taking Advantage of the Phage-Bacterium Coevolutionary Biology
3.4 Designing Effective Phages by Engineering
4 Conclusions
References
Chapter 24: Bacteriophage Virus-Like Particles: Platforms for Vaccine Design
1 Introduction
2 Bacteriophage VLPs and Their Applications
2.1 MS2 VLPs
2.2 PP7 VLPs
2.3 Qฮฒ VLPs
2.4 AP205 VLPs
2.5 P22 VLPs
2.6 Mutant T4 Phage
3 Antigen Display Approaches on VLPs
3.1 Genetic Insertion
3.2 Chemical or Biological Conjugation
4 Conclusions
References
Index