Preface
Contents
Contributors
Part I: Isolation, and Characterization of Bacteriophages
Chapter 1: Isolation of Bacteriophages for Clinically Relevant Bacteria
1 Introduction
2 Materials
2.1 Enrichment of the Sample for Bacteriophage Isolation
2.2 Checking for the Presence of Bacteriophages in the Enriched Samples
2.3 Isolation of all Different Bacteriophages Based on Plaque Characteristics from the Enriched Samples
3 Methods
3.1 Enrichment of the Bacteriophage Isolation Source
3.1.1 Using Liquid Samples
3.1.2 Using Solid Samples
3.2 Checking for the Presence of Bacteriophages in the Enriched Samples
3.2.1 Spot Test Verification of the Enriched Samples
3.2.2 Paper Strip Spreading Method
3.2.3 Plating the Enriched Sample
3.3 Isolation of All Different Bacteriophages Based on Plaque Characteristics from the Enriched Samples
4 Notes
References
Chapter 2: Observation of Bacteriophage Ultrastructure by Cryo-Electron Microscopy
1 Introduction
2 Materials
2.1 Support Preparation for Negative Staining
2.2 Fast Freezing
2.3 Data Acquisition
3 Methods
3.1 Negative Staining
3.2 Fast Freezing
3.3 Data Acquisition
3.4 High-Resolution Structure Determination
3.4.1 Workflow
3.4.2 Processing Software
3.5 Structure Determination and the Use of Hybrid Methods
4 Notes
References
Chapter 3: Bacteriophage Taxonomy: A Continually Evolving Discipline
1 Why Is Taxonomy Important?
2 Brief History of Phage Taxonomy Prior to 2008
2.1 Extension of Proteomics to Phage Taxonomy from 2008
2.2 DNA Sequence Comparisons Enter the Picture
3 How ICTV Currently Groups Phages into Taxa
3.1 Progress
3.2 Taxonomic Challenges of Modern Sequence Databases
3.3 Creation of Higher Taxa
4 Concluding Statement
5 Practical Considerations for Phage Scientists
References
Part II: Selection, Production, and Encapsulation of Bacteriophages
Chapter 4: Guidelines to Compose an Ideal Bacteriophage Cocktail
1 Introduction
1.1 Selection of Bacterial Host Strains
1.1.1 Bacterial Strains Used for the Selection and Initial Propagation of Therapeutic Bacteriophages in the Preproduction Proc...
1.1.2 Bacterial Strains Used for the Propagation of Bacteriophages in the Production Process
1.2 Selection of Candidate Bacteriophages and Design of a Therapeutic Cocktail
1.2.1 Recommended Isolation Sources for Therapeutic Bacteriophages
1.2.2 Requirements for Single Therapeutic Bacteriophage Candidates
1.2.3 Phages in Cocktails
2 Materials
2.1 Modified Appelmans Method for the Preadaptation of Phages Against Bacterial Strains and the Evaluation of Phage Complement...
2.2 Modified Appelmans´ Method for the Preadaptation of Phages on Bacterial Strains Using the Omnilog System
2.3 Evaluation of Complementary Activity of Phages in the Cocktail with the Omnilog System
3 Methods
3.1 Modified Appelmans Method for the Preadaptation of Phages Against Bacterial Strains and the Evaluation of Phage Complement...
3.2 Modified Appelmans´ Method for the Preadaptation of Phages on Bacterial Strains Using the Omnilog System
3.3 Evaluation of Complementary Activity of Phages in the Cocktail with the Omnilog System
4 Notes
References
Chapter 5: Rapid Bench to Bedside Therapeutic Bacteriophage Production
1 Introduction
2 Materials
2.1 Equipment
2.2 Bacteriophage and Bacterial Host Strains
2.3 Reagents
2.4 Consumables
2.5 Recommended Kits
3 Methods
3.1 Preprocessing
3.1.1 Bacterial Host Standard Curve
3.1.2 Spot+ Phage Titration
3.1.3 Pilot Phage Amplification
3.1.4 Lysate Prophage Detection
3.2 Batch Phage Amplification
3.3 Phage Purification
3.3.1 Dead-End Filtration Sterilization
3.3.2 Ultrafiltration and Diafiltration
3.3.3 Chromatographic Removal of Endotoxin
3.3.4 Free Nucleic Acids Digestion
3.4 Final Preparation Validation
3.4.1 Sterilization and Titration
3.4.2 Endotoxin Quantification
3.5 Sterile Fill-Finish
4 Notes
References
Chapter 6: Bacteriophage Production in Compliance with Regulatory Requirements
1 Introduction
2 Regulatory Requirements
3 Production Processes
3.1 Bacteriophage Propagation
3.2 Bacteriophage Purification
4 Quality, Safety, and Efficacy Requirements for Bacteriophage Products
5 Conservation, Storage, and Stability
References
Chapter 7: Nano/microformulations for Bacteriophage Delivery
1 Introduction
2 Materials
2.1 Lipidic Encapsulation
2.2 Alginate/CaCO3 Encapsulation
2.3 Characterization of Nano/microparticles
2.4 Encapsulation Efficiency
2.5 Microscopy of Nano/microparticles
3 Methods
3.1 Lipidic Encapsulation
3.2 Alginate/CaCO3 Encapsulation
3.3 Characterization of Nano/microparticles
3.3.1 Lipidic Nanoparticles
3.3.2 Alginate/CaCO3 Microparticles
3.4 Calculation of Encapsulation Efficiency
3.4.1 Lipidic Nanoparticles
3.4.2 Alginate/CaCO3 Microparticles
3.5 Microscopy Observation
3.5.1 Lipidic Nanoparticles Cryo-TEM Microscopy
3.5.2 Alginate/CaCO3 Microparticles
4 Notes
References
Part III: From In Vitro to Animal Efficacy Studies
Chapter 8: Phage-Host Interaction Analysis Using Flow Cytometry
1 Introduction
2 Materials
2.1 Synchronized Bacteriophage Infection of Planktonic Cultures
2.2 Flow Cytometry Analysis
3 Methods
3.1 Synchronized Bacteriophage Infection of Planktonic Cultures
3.2 Flow Cytometry Analysis
4 Notes
References
Chapter 9: Bacteriophage Control of Infectious Biofilms
1 Introduction
2 Materials
2.1 Biofilm Formation
2.2 Biofilm Control with Bacteriophages
2.3 Evaluation of Biofilm Treatment with Bacteriophages
2.3.1 Quantification of Biofilm Culturable Cells
2.3.2 Quantification of the Biofilm Biomass
2.3.3 Bacteriophage Titration by Drop Plaque Assay
3 Methods
3.1 Biofilm Formation
3.2 Biofilm Control with Bacteriophages
3.3 Evaluation of Biofilm Treatment with Bacteriophages
3.3.1 Quantification of Biofilm Culturable Cells
3.3.2 Quantification of Biofilm Biomass
3.3.3 Bacteriophage Titration by Drop Plaque Assay
4 Notes
References
Chapter 10: Studying Bacteriophage Efficacy Using a Zebrafish Model
1 Introduction
2 Materials
2.1 GFP+-PAO1 Culture Preparation
2.2 High-Titer Bacteriophage Stocks Preparation and Purification
2.3 Zebrafish (Danio rerio) Breeding and Embryos Collection
2.4 Bacterial Infection and Phage Treatment
2.5 Outcomes
3 Methods
3.1 GFP+-PAO1 Culture Preparation
3.1.1 Preparation of the Bacterial Inoculum for In Vivo Experiments
3.2 High-Titer Bacteriophage Stocks Preparation and Purification
3.2.1 Preparation of the Phage Suspension for In Vivo Experiments
3.3 Zebrafish (Danio rerio) Breeding and Embryos Collection
3.4 Bacterial Infection and Phage Treatment
3.4.1 Injection of Bacterial Suspensions in Embryos
3.4.2 Selection of Locally Injected GFP+ PAO1 Infected Embryos
3.4.3 Injection of Bacteriophage Suspensions in Embryos
3.4.4 Time-Lapse Confocal Microscope Imaging of Phage Therapy in Locally/Systemically Infected Embryos
3.5 Outcomes
3.5.1 Evaluation of the Severity of Bacterial Infection
3.5.2 Determination of Bacterial Burden by CFU Counts
3.5.3 Determination of Bacterial Load by Fluorescent Pixel Count (FPC)
4 Notes
References
Chapter 11: Use of Galleria mellonella as an Animal Model for Studying the Antimicrobial Activity of Bacteriophages with Poten...
1 Introduction
2 Materials
2.1 Strains of Bacteria and Types of Bacteriophages
2.2 Culture Media and Buffers
2.3 Equipment
3 Methods
3.1 Preparation of Bacteria
3.2 Bacteriophage Preparation
3.3 Preparation of G. mellonella Larvae
3.4 G. mellonella Infection: Testing the Lethal Dose (LD50)
3.5 G. mellonella Infection and Treatment with Bacteriophages
3.6 Survival Analysis
3.7 Extraction of Larval Hemolymph for Quantification of Bacteria and Bacteriophages
4 Notes
References
Part IV: Host Interaction and Clinical Application
Chapter 12: Interaction of Bacteriophages with the Immune System: Induction of Bacteriophage-Specific Antibodies
1 Introduction
2 Materials
2.1 Preparation of Bacteriophages or Bacteriophage Proteins
2.2 Immunization of Mice
2.3 Testing Specific Antibody Levels in the Blood
3 Methods
3.1 Preparation of Bacteriophage or Bacteriophage Proteins
3.2 Immunization of Mice
3.3 Testing Specific Antibody Levels in Blood
4 Notes
References
Chapter 13: Bacteriophage Treatment of Infected Diabetic Foot Ulcers
1 Introduction
2 Materials
3 Methods
3.1 Microbial Strain Isolation and Identification
3.2 Selection of Specific Bacteriophage/Bacteriophages
3.3 Making Bacteriophage Preparation for Treatment
3.4 Bacteriophage Treatment
4 Notes
References
Chapter 14: A Review of Phage Therapy for Bone and Joint Infections
1 Introduction
2 Relevance of Phage Therapy to Treat BJI
2.1 Pathophysiology of BJI
2.2 The Double Action of Phages
3 High Variability of the Different Clinical Forms and Complexity of the Management of the Disease
3.1 All BJIs Are Not the Same Diseases
3.2 Management of Posttraumatic Infections
3.3 Prosthetic Joint Infections
3.4 BJIs Related to Step-by-Step Contamination of the Bone
3.5 Hematogenous Seeding BJIs
4 Guidelines for the Administration of Phages in Patients with BJIs
4.1 Historical Ways of Phage Administration
4.2 Ways of Administration in Contemporary Orthopedic Surgery
4.3 Immunization and Clearance of Phages Depending on the Way of Administration
4.4 The Use of Phages Is More or Less Relevant and Easy in the Field of BJI
4.5 Potential Nonrelevant and Relevant Indications of Phage Therapy to Treat BJIs
4.5.1 Nonrelevant Indications for Phage Therapy
4.5.2 Potential Indications for Phage Therapy
4.5.3 Relevant Indications and Strategies Currently Developed in the Clinic
5 Phage Therapy Experiences for BJI
5.1 BJIs Associated with Accessible Abscesses
5.2 Prosthetic Joint Infection
6 Conclusion and Perspective
References
Chapter 15: Successful Use of Phage and Antibiotics Therapy for the Eradication of Two Bacterial Pathogens from the Respirator...
1 Introduction
2 Patient
3 Bacterial Isolates
4 Bacteriophages
5 Results and Discussion
6 Conclusion
References
Part V: Phage Engineering Approaches
Chapter 16: Genetic Engineering and Rebooting of Bacteriophages in L-Form Bacteria
1 Introduction
2 Materials
2.1 Antibiotic-Induced L-Form Conversion
2.2 DNA Synthesis and Assembly
2.3 L-Form Rebooting and Phage Recovery
3 Methods
3.1 Antibiotic-Induced L-Form Conversion
3.2 DNA Synthesis and Assembly
3.3 L-Form Rebooting
3.4 Phage Recovery
4 Notes
References
Chapter 17: Synthetic Biology to Engineer Bacteriophage Genomes
1 Introduction
2 Materials
2.1 Bacteriophage Recombineering of Electroporated DNA (BRED)
2.1.1 Preparation of Recombineering Cells
2.1.2 Bred
2.1.3 Recovery and Confirmation of Mutant Bacteriophages
2.2 Yeast-Based Assembly of Bacteriophage Genomes
2.2.1 Bacteriophage DNA Isolation
2.2.2 Preparation of Yeast Competent Cells
2.2.3 Yeast Transformation
2.2.4 Yeast Colony PCR
2.2.5 Plaque Formation Assays
3 Methods
3.1 Bacteriophage Recombineering of Electroporated DNA
3.1.1 Preparation of Recombineering Competent Cells
3.1.2 Recombineering of Bacteriophage DNA
3.1.3 Recovery and Confirmation of Mutant Bacteriophages
3.2 Yeast-Based Assembly of Bacteriophage Genomes
3.2.1 Bacteriophage DNA Isolation (See Note 23)
3.2.2 Preparation of Yeast-Competent Cells
3.2.3 Yeast Transformation
3.2.4 Yeast Colony PCR to Check for the Correct DNA Assembly
3.2.5 Plaque Formation Assays
4 Notes
References
Chapter 18: Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems
1 Introduction
2 Materials
2.1 Strains
2.2 Growth Media and Reagents
2.3 Plasmid Construction via PCR
2.4 Phage Genomic DNA Extraction
3 Methods
3.1 Select a Functional Protospacer
3.2 Construct the Targeting Strain
3.3 Confirm Functionality of the Targeting Strain
3.4 Construct the Editing Strain
3.5 Utilize the Editing Strain to Generate Phage Recombinants
3.6 Purify and Confirm Phage Recombinants
3.6.1 Purify Putative Phage Recombinants
3.6.2 Prepare High-Titer Phage Lysates
3.6.3 Extract Genomic DNA from High-Titer Phage Lysates
3.6.4 Sequence Phage DNA
3.7 Introducing Mutations Distal to the Targeted Region
3.8 Creating Deletions in a Single Step
3.9 Editing Phages that Infect CRISPR-Less Hosts
3.10 Preparing Electrocompetent Staphylococci
3.11 Electroporating Competent Staphylococci
4 Notes
References
Chapter 19: Robust and Reproducible Protocol for Phage Genome Rebooting´´ Using Transformation-Associated Recombination (TAR...
1 Introduction
2 Materials
2.1 Incorporation of the Phage Genome into Yeast Centromeric Plasmid (TAR Cloning)
2.2 Isolation of a Yeast Centromeric Plasmid Containing the Bacteriophage Genome
2.3 Preparation of Electrocompetent E. Coli Cells
2.4Rebooting´´ of the T7 Bacteriophage Genome in Its Natural Host (E. coli)
2.5 Rebooting´´ the Genome of Bacteriophage KP32_192 Specific to K. pneumoniae Using an Intermediate E. coli Host
3 Methods
3.1 Incorporation of the Phage Genome into a Yeast Centromeric Plasmid (TAR Cloning)
3.1.1 Obtaining a PCR Copy of Yeast Plasmid pRSII415 Flanked by 5′- and 3′-Terminal Sequences of the Phage T7 Genome
3.1.2 Integration of Phage Genomic DNA into Yeast Plasmid Backbone (TAR Cloning)
3.1.3 Yeast Colony Screening
3.2 Isolation of a Yeast Centromeric Plasmid Containing the Bacteriophage Genome
3.3 Preparation of Electrocompetent E. coli Cells
3.4Rebooting´´ of the T7 Bacteriophage Genome in Its Natural Host (E. coli)
3.5 ``Rebooting´´ the Genome of Bacteriophage KP32_192 Specific to K. pneumoniae Using an Intermediate E. coli Host
4 Notes
References
Index