Clostridium difficile: Methods and Protocols (Methods in Molecular Biology, 1476)

✍ Scribed by Adam P. Roberts (editor), Peter Mullany (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 292
• Category: Library

✦ Synopsis

This second edition provides 21 new chapters on methods used in laboratories for investigating the physiology and molecular genetics of the pathogen Clostridium difficile. Chapters detail up-to -date experimental techniques for gene editing and transcriptional analysis which are used to investigate the fundamental biology of the organism and its virulence factors. Additional chapters describe development of potential new treatments including vaccines, bacteriophage and faecal transplantation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Clostridium difficile: Methods and Protocols, Second Edition provides a comprehensive catalogue of molecular tools and techniques authored by the researchers who have developed them.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates
1 Introduction
2 Materials
2.1 Selective Media Components
2.2 Nonselective Media Components
2.3 DNA Isolation Reagents
2.4 HindIII Restriction Enzyme Solution
2.5 Horizontal Agarose Gel Electrophoresis Components
3 Methods
3.1 Clostridium difficile (CD) Recovery and Purification from Stool Specimens by Sequential Selective (TCCFA) and Non selective (Anaerobic Sheep’s Blood Agar) Plate Culture
3.2 Isolation of Whole Cellular CD DNA
3.3 HindIII Restriction of Purified Whole Cellular C. difficile DNA
3.4 Horizontal Agarose Gel Electrophoresis of HindIII-­Restricted CD DNA
3.5 Visual Analysis of Restriction Patterns on REA Gel Photographs
4 Notes
References
Chapter 2: Direct PCR-Ribotyping of Clostridium difficile
1 Introduction
2 Materials
2.1 Total Stool DNA Extraction
2.2 PCR Amplification
2.3 Agarose Gel Electrophoresis
2.4 Analysis of Banding Patterns
3 Method
3.1 Isolation of Total Stool DNA
3.2 PCR Amplification
3.3 Agarose Gel Electrophoresis
3.4 Analysis of Banding Patterns and Determination of PCR-Ribotypes
4 Notes
References
Chapter 3: From FASTQ to Function: In Silico Methods for Processing Next-Generation Sequencing Data
1 Introduction
2 Materials
2.1 Software
2.2 Preparing the System
2.3 Worked Example Data
3 Methods
3.1 QC the Data
3.2 Assemble
3.3 Order Contigs
3.4 Annotation of Contigs
3.5 Compare to Reference
3.6 Identify Multi-­Locus Strain Type (MLST) In Silico
4 Notes
References
Chapter 4: Clostridium difficile Genome Editing Using pyrE Alleles
1 Introduction
2 Materials
2.1 Culture Media
2.2 Antibiotic and Other Supplements
2.3 Molecular Biology Reagents, Strains, and Plasmids
3 Methods
3.1 Design of KO Cassette and Incorporation into the KO Vector
3.2 Conjugation and Isolation of Single-­Crossover Clones
3.3 Isolation of Double-­Crossover Clones
3.4 Correction Back to pyrE+
3.5 Complemen
4 Notes
References
Chapter 5: Use of mCherryOpt Fluorescent Protein in Clostridium difficile
1 Introduction
2 Materials
2.1 Plasmid Constructions in Escherichia coli
2.2 Transfer of pDSW1728 or pRAN473 Derivatives to C. difficile by Conjugation
2.3 Growth of C. difficile Exconjugants for Studies of Gene Expression and Protein Localization Using mCherryOpt Plasmid Constructs
2.4 Fixation Reagents
2.5 Detection and Quantification of mCherryOpt in C. difficile
2.5.1 Fluorescence Microscopy
2.5.2 Fluorometer (Fluorescent Plate Reader)
2.5.3 Flow Cytometry
3 Methods
3.1 Guidelines for Constructing mCherryOpt Promoter Fusions
3.2 Guidelines for Constructing mCherryOpt Gene Fusions
3.3 Conjugation
3.4 Growth of  C. difficile/pDSW1728 Derivatives to Monitor Gene Expression
3.5 Growth of C. difficile/pRAN473 Derivatives to Monitor Protein Localization
3.6 Fixation
3.7 Analysis by Fluorescence Microscopy
3.8 Analysis Using a Fluorometer (Fluorescent Plate Reader)
3.9 Analysis by Flow Cytometry
3.10 Critical Parameters and Troubleshooting
4 Notes
References
Chapter 6: A Fluorescent Reporter for Single Cell Analysis of Gene Expression in Clostridium difficile
1 Introduction
2 Materials
2.1 Culture Media
2.2 Fluorescent Dyes
2.3 Solutions for Microscopy
2.4 SNAP and CLIP Substrates
2.5 SDS-­Polyacrylamide Gels and Electroblotting to Nitrocellulose Membranes
2.6 Antibodies
3 Methods
3.1 SNAP/CLIP Labeling for Microscopy
3.2 SNAP/CLIP Labeling for Flow Cytometry Analysis
3.3 SNAP/CLIP-Tag Fusions Detected by In-Gel Fluorescence
3.4 Western Blot Analysis
4 Notes
References
Chapter 7: Clostridium difficile Adhesins
1 Introduction
2 Materials
2.1 Bacterial Culture Media
2.2 In Vitro Adhesion and Inhibition Adhesion Assays
2.3 In Vivo Adhesion Assay
2.4 Immunoelectron Microscopy
2.5 Far-Immuno Dot Blotting
2.6 Indirect Immunofluorescence
2.7 Enzyme-Linked Immunosorbent Assay (ELISA)
2.8 C. difficile Strains
3 Methods
3.1 In Vitro Adhesion and Inhibition Adhesion Assays
3.1.1 Cell Lines: Caco-2 TC7, Vero Cells
3.1.2 In Vitro Adherence Assay
3.1.3 In Vitro Adherence Inhibition Assays
Inhibition by Specific Antibodies
Inhibition by Recombinant Proteins
3.2 In Vivo Adherence with a Monoxenic C. difficile Mouse Model
3.3 Immunoelectron Microscopy to Visualize the Surface Localization of a Protein of Interest
3.4 Binding of FbpA to Extracellular Matrix Proteins by Far-Immuno Dot-Blotting
3.5 C. difficile Binding to Soluble Fibronectin by Indirect Immunofluorescence
3.6 C. difficile Binding to Immobilized Fibronectin by ELISA
4 Notes
References
Chapter 8: Intestinal Epithelial Cell Response to Clostridium difficile Flagella
1 Introduction
2 Materials
2.1 Purification of C. difficile Recombinant Flagellin
2.2 Cell Culture
2.3 Protein Extraction from Cell Cultures
2.4 SDS PAGE
2.5 Western Immunoblotting
2.6 ELISA
3 Methods
3.1 Purification of  C. difficile Recombinant Flagellin
3.2 Cell Culture
3.3 Stimulation of Caco-2 Cells with FliC
3.4 Protein Extraction from Cell Cultures
3.5 SDS PAGE
3.6 Western Immunoblotting
3.7 ELISA-Based Quantitative Array
4 Notes
References
Chapter 9: Isolating and Purifying Clostridium difficile Spores
1 Introduction
2 Materials
2.1 Culture Media
2.2 Media Supplements
2.3 Solutions for  C. difficile Spore Stocks
2.4 Additional Reagents
3 Methods
3.1 Growth and Sporulation Efficiency of C. difficile on Various Media
3.2 Isolation of  C. difficile Spores from Solid Medium
3.3 Purification of  C. difficile Spores
3.4 Enumeration of Initial Spore Stock and Serial Dilutions
3.5 Preparation of Final Working Spore Stocks
4 Notes
References
Chapter 10: Inducing and Quantifying Clostridium difficile Spore Formation
1 Introduction
2 Materials
2.1 Reagents
2.2 Media
2.3 Equipment
3 Methods
3.1 Inducing Sporulation
3.2 Quantifying Sporulation Using Heat Resistance
4 Notes
References
Chapter 11: Characterization of Functional Prophages in Clostridium difficile
1 Introduction
1.1 Identification of Prophages in Bacterial Genomes
1.2 Prophage Induction
1.3 Identification of a Propagating Host
1.4 Observation of Phage Particles by Transmission Electron Microscope (TEM)
1.5 Whole Phage Genome Analyses
1.6 Isolation of Bacterial Lysogens Carrying a Newly Identified Phage
2 Materials
2.1 Mitomycin C Prophage Induction in Broth
2.2 UV Prophage Induction in Broth and on Agar Plates
2.3 Phage Propagation in Broth and Soft Agar
2.4 Observation of Phages and Lysates by Transmission Electron Microscopy
2.5 Extraction of Phage DNA (In-House Mini-Prep Protocol)
3 Methods
3.1 Mitomycin C Prophage Induction in Liquid Broth
3.2 UV Prophage Induction in Liquid Broth
3.3 Identification of a Susceptible Host for Phage Propagation
3.4 UV Prophage Induction with Simultaneous Identification of a Propagating Host
3.5 Phage Purification by Plaque Assay
3.6 Phage Propagation: First Amplification
3.7 Phage Propagation: Second Amplification
3.8 Observation of Phage Particles Under the Electron Microscope
3.9 Extraction of Phage DNA for Whole Genome Analyses
3.10 Analysis of Phage Genomes by REA
3.11 Creation of New Lysogens
4 Notes
References
Chapter 12: Induction and Purification of C. difficile Phage Tail-Like Particles
1 Introduction
2 Materials
2.1 C. difficile Growth and PTLP Induction
2.2 PTLP Precipitation
2.3 Electron Microscopy
2.4 CsCl Purification
2.5 Concentration
3 Methods
3.1 C. difficile Growth
3.2 PTLP Induction
3.3 PTLP Precipitation
3.4 Cesium Chloride Gradient Purification
3.5 Concentration of PTLP Samples
3.6 Electron Microscopy
4 Notes
References
Chapter 13: Phage Transduction
1 Introduction
2 Materials
2.1 Bacterial Growth and Phage Propagation Media
2.2 Broth Microdilution MIC Susceptibility Assays
2.3 Phage Purification
2.4 PCR
2.5 Transduction
3 Methods
3.1 Finding Donors and Recipients of Antibiotic Resistant Genes (ARG) for Phage Transduction
3.2 PCR of Antibiotic Resistant Donor Isolates
3.3 Propagate Phage in Donor (Agar Overlay Method)
3.4 Semi-purify Crude Phage Suspension
3.5 Determine Phage Titer
3.6 Transduction
3.7 Validation of Transductants
4 Notes
References
Chapter 14: Transfer of Clostridium difficile Genetic Elements Conferring Resistance to Macrolide–Lincosamide–Streptogramin B (MLSB) Antibiotics
1 Introduction
2 Materials
2.1 Culture and Isolation
2.2 Minimal Inhibitory Concentrations (MICs) Determination
2.3 Filter Mating Assay
2.4 Standard Molecular Biology Procedures
2.4.1 PCR Amplification of the erm (B) Gene
2.4.2 Molecular Characterization of erm(B)-Containing Elements by PCR-Mapping
2.4.3 PCR-Ribotyping
2.4.4 Gel Electrophoresis
2.5 Southern Blotting
3 Methods
3.1 MICs Determination of Erythromycin, Clindamycin, and Rifampin Using Etest Strip
3.2 erm(B) Detection
3.3 Determination of the erm(B)-Containing Element Genetic Organization
3.4 Selection of Rifampin—Resistant Colonies
3.5 Filer Mating Assay
3.6 PCR-Ribotyping
3.7 Southern Blotting
3.7.1 DNA Hybridization
Probe Synthesis
Transfer of DNA
3.7.2 Hybridization
3.7.3 Detection
4 Notes
References
Chapter 15: Methods for Determining Transfer of Mobile Genetic Elements in Clostridium difficile
1 Introduction
2 Materials
2.1 Detection of Excision of Elements from the Genome
2.2 Determination of Transfer: Filter and Plate Matings
2.3 Analysis of  C. difficile Transconjugants  by PCR
2.4 Analysis of  C. difficile Transconjugants by Southern Blot
3 Methods
3.1 Detection of Exciton of Elements from the Genome
3.2 Determination of Transfer
3.2.1 Filter Mating Transfer of Tn5398 from 630 to CD37
3.2.2 Plate Matings: Transfer of Tn4453 from JIR8156 (630:: Tn4453) to CD37
3.3 Confirmation of Transconjugants by PCR
3.4 Confirmation of Transconjugants by Southern Hybridization
4 Notes
References
Chapter 16: Investigating Transfer of Large Chromosomal Regions Containing the Pathogenicity Locus Between Clostridium difficile Strains
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Growth Media
2.3 Transfer via Conjugation
2.4 Confirmation of Transconjugants
2.5 Mitomycin C Induction and Phage Infection
2.6 Next-Generation Sequencing (See Note 5)
3 Methods
3.1 Transfer via  Conjugation
3.2 Confirmation of Transconjugants
3.3 Mitomycin C Induction and Phage Infection
3.4 Next-Generation Sequencing
4 Notes
References
Chapter 17: An In Vitro Model of the Human Colon: Studies of Intestinal Biofilms and Clostridium difficile Infection
1 Introduction
2 Materials
2.1 Traditional Chemostat Vessel
2.2 Biofilm Chemostat Vessel
2.3 Growth Medium
2.4 Solid Agar Medium
2.5 Cytotoxin Assay
3 Methods
3.1 Vessel Assembly
3.2 Preparation of the Gut Model
3.3 Preparation of Fecal Emulsion
3.4 C. difficile Spore Preparation
3.5 Planktonic Bacterial Population Sampling and Enumeration
3.6 Biofilm Analysis
3.7 Cytotoxin Assay
3.8 Reduced Antimicrobial Susceptibility Surveillance
3.9 Antimicrobial Bioassay
3.10 Experimental Design
3.10.1 Induction Gut Model
3.10.2 Treatment Gut Model
4 Notes
References
Chapter 18: MiniBioReactor Arrays (MBRAs) as a Tool for Studying C. difficile Physiology in the Presence of a Complex Community
1 Introduction
2 Materials
2.1 Fecal Sample Processing for −80 °C Storage
2.2 MBRA Assembly and Operation
2.3 MBRA and  C. difficile Cultivation Media (See Note 1)
2.4 MBRA Inoculation and Sampling
2.5 qPCR to Enumerate C. difficile
3 Methods
3.1 Fecal Sample Preparation for − 80 °C Storage
3.2 MBRA Assembly for 24 Reactors (Four MBRA Strips)
3.2.1 MBRA Source Bottle Assembly and Media Preparation
3.3 MBRA Setup
3.4 MBRA Inoculation
3.5 MBRA Operation and C. difficile Invasion
3.6 Alternative Bioreactor Assembly
3.7 Quantification of C. difficile Levels by qPCR
4 Notes
References
Chapter 19: A Practical Method for Preparation of Fecal Microbiota Transplantation
1 Introduction
2 Materials
2.1 Patient and Donor Selection
2.2 Processing of Feces
3 Methods
3.1 Donor Selection and Screening
3.2 Processing of Feces
4 Notes
References
Chapter 20: Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine
1 Introduction
2 Materials
2.1 Chemical Reagents
2.2 Equipment
3 Methods
3.1 Solution Preparation
3.1.1 Mobile Phase A (2 L)
3.1.2 Mobile Phase B (2 L)
3.1.3 Storage Solution (1 L)
3.2 Sample Preparation
3.3 IEX HPLC
4 Notes
References
Chapter 21: A Size-Exclusion Chromatography Method for Analysis of Clostridium difficile Vaccine Toxins
1 Introduction
2 Materials
2.1 Chemicals and Reagents
2.2 Equipment
3 Methods
3.1 Solution Preparation
3.2 Sample Preparation
3.3 SEC HPLC
4 Notes
References
Index