Bacterial Persistence: Methods and Protocols (Methods in Molecular Biology, 2357)

✍ Scribed by Natalie Verstraeten (editor), Jan Michiels (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 292
• Edition: 2nd ed. 2021
• Category: Library

✦ Synopsis

This volume presents a collection of methods that have contributed to the current understanding of bacterial persisters. Chapters in the book detail general guidelines for measuring persister levels in bacterialcultures, strategies to enrich and resuscitate persister subpopulations, single-cell approaches for visualizing and characterizing persisters, omics techniques and cellular and animal models for studying persistence. 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, Bacterial Persistence: Methods and Protocols, Second Edition aims to be a useful practical guide to researchers to help further their study in this field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Introduction
Chapter 1: Studying Bacterial Persistence: Established Methods and Current Advances
1 Bacterial Persistence
2 Clinical Relevance
3 Challenges in Persistence Research
4 Current Methods
4.1 Genetic Screenings
4.2 Experimental Evolution
4.3 Transcriptomics and Proteomics
4.3.1 Transcriptomics
4.3.2 Proteomics
4.4 Single-cell Techniques
4.4.1 Single-cell Time-Lapse Microscopy
4.4.2 Flow Cytometry
4.4.3 Advances in Single-cell Technologies
4.5 Mathematical Modelling
4.6 In vivo Models
5 Future Perspectives
References
Part II: Quantification and Enrichment of Persister Cells
Chapter 2: Antibiotic Tolerance and Persistence Studied Throughout Bacterial Growth Phases
1 Introduction
2 Materials
3 Methods
3.1 Determination of Antibiotic Tolerance Along the Growth Curve
3.1.1 Day 0: Collection of Materials and Inoculation of the Overnight Culture
3.1.2 Day 1: Subculturing
3.1.3 Day 2: Antibiotic Tolerance Assay
3.1.4 Day 3: CFU Counting
3.1.5 Day 4: CFU Counting and Plotting
3.2 Determination of Antibiotic Tolerance in Stationary Phase
3.2.1 Day 0: Preparation of Media and Cultures
3.2.2 Day 1: Subculturing
3.2.3 Day 3: Stationary-Phase Antibiotic Tolerance Assay
3.2.4 Day 4: CFU Counting
3.2.5 Day 5: CFU Counting and Plotting
4 Notes
References
Chapter 3: A Robust Method for Generating, Quantifying, and Testing Large Numbers of Escherichia coli Persisters
1 Introduction
2 Materials
2.1 Generation of Large Fractions of Tolerant Cells
2.2 Identification and Quantification of Tolerant Cells Using Membrane Staining and Flow Cytometry
2.3 Assessment of Antibiotic Tolerance with Flow Cytometry
3 Methods
3.1 Generation of Large Fractions of Tolerant Cells
3.2 Identification and Quantification of Tolerant Cells Using Membrane Staining and Flow Cytometry
3.3 Guide on How to Use the Matlab Script
3.4 Assessment of Antibiotic Tolerance with Flow Cytometry
4 Notes
References
Chapter 4: Enrichment of Persister Cells Through -Lactam-Induced Filamentation and Size Separation
1 Introduction
2 Materials
3 Methods
3.1 Installation of the Vacuum Filtration System
3.2 Persister Enrichment Protocol
4 Notes
References
Chapter 5: Methods for Enrichment of Bacterial Persister Populations for Phenotypic Screens and Genomic Studies
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Exponentially Dividing Bacterial Cultures
3.2 Determination of Minimal Bactericidal Antibiotic Concentration
3.3 Enrichment Method #1: Flow Sorting for Persisters by Staining Membrane Depolarization
3.4 Enrichment Method #2: Antibiotic Lysis of Nonpersister Cells
3.5 Enrichment Method #3: Culture Aging to Induce Persister Cell Survival
3.6 Bacterial Cell Lysis with Lysozyme Pretreatment
3.7 Bacterial RNA Extraction
4 Notes
References
Part III: Single-Cell Analysis of Persister Cells
Chapter 6: Observing Bacterial Persistence at Single-Cell Resolution
1 Introduction
2 Materials
2.1 Determining Time Delay in Killing of Growing Cells
2.2 Single-Cell Observation of Lag Time After Starvation
3 Methods
3.1 Determining Time Delay in Killing of Growing Cells
3.2 Single-Cell Observation of Lag Time After Starvation
3.3 Preparing the Agarose Pad
3.4 Sandwiching Cells
3.5 Microscopy
3.6 MicrobeJ Analysis
4 Notes
References
Chapter 7: Phenotypic Characterization of Antibiotic Persisters at the Single-Cell Level: From Data Acquisition to Data Analys...
1 Introduction
2 Materials
3 Methods
3.1 Data Acquisition
3.1.1 Day 1: Preparation of Cultures and Medium
3.1.2 Day 2: Precultures
3.1.3 Day 3: Cultures and Microscopy
3.2 Data Analysis
3.2.1 Cell Segmentation, Cell Tracking and Intensity/Morphology Measurements
3.2.2 Defining and Monitoring Key Parameters for Persistence
3.2.3 Persister Identification
3.2.4 Microcolony Growth Rate
3.2.5 Single-Cell Elongation Rate
3.2.6 Persisters Growth Stage Identification
3.2.7 Fluorescent Biosensors
4 Notes
References
Chapter 8: Microfluidics for Single-Cell Study of Antibiotic Tolerance and Persistence Induced by Nutrient Limitation
1 Introduction
2 Materials
2.1 Bacterial Strains and Growth Media
2.2 Preparation of Microfluidics Device
2.3 Cell Observation and Microfluidic Experiments
2.4 Additional Equipment for Setup with Temporal Heterogeneity in Nutrient Availability
3 Methods
3.1 Preparation of Microfluidics Devices
3.2 Setup with Spatial Heterogeneity in Nutrient Availability
3.3 Setup with Temporal Heterogeneity in Nutrient Availability
4 Notes
References
Chapter 9: Counting Chromosomes in Individual Bacteria to Quantify Their Impacts on Persistence
1 Introduction
2 Materials
2.1 Bacterial Strains and Plasmids
2.2 Media
2.3 Nucleic Acid Staining
2.4 Persister Assay
2.5 Fluorescence Activated Cell Sorting (FACS)
2.6 Microscopy
3 Methods
3.1 Cell Growth Conditions
3.2 Nucleic Acid Staining Selection
3.2.1 Hoechst 33342 Staining Procedure
3.2.2 Determination of the Impact of Hoechst 33342 Staining on Culturability and Persistence
3.3 Sample Preparation for Fluorescence Activated Cell Sorting (FACS)
3.3.1 Cell Dilution and Staining for FACS
3.3.2 Other Preparations for FACS
3.4 Controls for FACS: Determination of Whether the Timing Required to Sort Cells Using FACS Influences Persistence
3.5 FACS
3.6 Verification of Sorting Fidelity Based on DNA Content
3.6.1 Confirmation of DNA Content of Sorted Cells Using a Secondary dsDNA-Specific Dye (PicoGreen)
3.6.2 Confirmation of DNA Content of Sorted Cells Using an Origin-of-Replication Reporter
3.6.3 Controls for Origin Reporter
3.6.4 Confocal Microscopy
3.7 Culturability and Persistence Assays on Cell Samples
3.7.1 Culturability and Persistence Assay on Sorted Fractions
3.7.2 Culturability and Persistence Assays on Unsorted Cells Diluted to the Same Density of Sorted Cells
4 Notes
References
Part IV: Omics Approaches in Persistence Research
Chapter 10: Analyzing Persister Proteomes with SILAC and Label-Free Methods
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Media and Reagents
2.2.1 Protein Sample Preparation
3 Methods
3.1 Pulsed-SILAC of Persister Cells
3.2 Persister Recovery
3.3 Sample Preparation for MS
3.4 MS Analysis
4 Notes
References
Chapter 11: Defining Proteomic Signatures to Predict Multidrug Persistence in Pseudomonas aeruginosa
1 Introduction
2 Materials
2.1 General Materials
2.2 Whole Cell Lysis and Protein Extraction
2.3 Sample Preparation for LC-MS/MS: Protein Digestion
2.4 Sample Preparation for LC-MS/MS: Solid-Phase Peptide Purification
2.5 LC-MS/MS
3 Methods
3.1 Sample Generation
3.2 Whole Cell Lysis and Protein Extraction
3.3 Sample Preparation for LC-MS/MS: Protein Digestion
3.4 Sample Preparation for LC-MS/MS: Solid-Phase Peptide Purification
3.5 LC-MS/MS Analysis
3.6 Peptide Level Data Set Generation
3.7 Regression and Feature Reduction of Proteomic Models of Persistence
3.7.1 Dataset Preparation
3.7.2 Orthogonal Partial Least Square Regression (oplsr) of MS Data
4 Notes
References
Chapter 12: The Use of Experimental Evolution to Study the Response of Pseudomonas aeruginosa to Single or Double Antibiotic T...
1 Introduction
2 Materials
2.1 General Materials
2.2 In Vitro Evolution Experiment
2.3 Determination of MIC
2.4 Genomic DNA Preparation
2.5 Isolation of Hypertolerant or Drug Resistant Strains
3 Methods
3.1 Evolution Experiment
3.2 MIC Determination
3.3 Preparation of gDNA and Genome Sequencing
3.3.1 Alignment to a Reference Genome
3.3.2 Call, Selection, and Quantification of Emerging Alleles
3.3.3 Identification of Regions of Recombination
3.4 Isolation of Individual Highly Tolerant or Drug Resistant Strains
3.5 Analysis of the Antibiotic-Related Characteristics of Individual Isolates
3.6 The Contribution of Low-Level Resistance to Survival and Tolerance
4 Notes
References
Part V: Persister Cell Resuscitation and Eradication
Chapter 13: Detecting Persister Awakening Determinants
1 Introduction
2 Materials
3 Methods
3.1 Identification of Persister Awakening Genes by FACS
3.1.1 Persister Assay and Control Samples
3.1.2 Preparatory Work
3.1.3 Sample Preparation
3.1.4 Sorting and Plating Metabolically Active Cells
3.1.5 Data Analysis
3.2 Identification of Persister Awakening Genes by Flow Cytometry
3.2.1 Persister Assay and Control Samples
3.2.2 Preparatory Work
3.2.3 Sample Preparation
3.2.4 Measuring the Number of Metabolically Active Cells
3.2.5 Plating Out the Samples
3.2.6 Data Analysis
3.3 Validation by Single-Cell Growth Dynamics
3.3.1 Sample Preparation
3.3.2 Visualization of Regrowth Using Time-Lapse Microscopy
3.3.3 Data Analysis
4 Notes
References
Chapter 14: Monitoring Persister Resuscitation with Flow Cytometry
1 Introduction
2 Materials
3 Methods
3.1 Preparing Overnight Precultures
3.2 Setting Up the Flow Cytometry Parameters
3.3 Beta-Lactam Mediated Cell Lysis
3.4 Clonogenic Survival Assay
3.5 Monitoring Persister Resuscitation
4 Notes
References
Chapter 15: Stimulating Aminoglycoside Uptake to Kill Staphylococcus aureus Persisters
1 Introduction
2 Materials
3 Methods
3.1 Quantifying Persister Cells
3.2 Determining Adjuvant Concentration
3.3 Evaluating Killing Against Chemically Induced Tolerant S. aureus
3.4 Texas Red-Conjugated Tobramycin Uptake
4 Notes
References
Part VI: Cellular and Animal Model Systems for Studying Persistence
Chapter 16: In Vitro Models for the Study of the Intracellular Activity of Antibiotics
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods
3.1 Preparation of Bacterial Suspension and of Media
3.2 Opsonization of Bacteria
3.3 Preparation of Eukaryotic Cells and Bacteria for Infection
3.4 Phagocytosis
3.5 Intracellular Growth
3.6 Assessment of Antibiotic Intracellular Activity
4 Notes
References
Chapter 17: Analysis of Salmonella Persister Population Sizes, Dynamics of Gut Luminal Seeding, and Plasmid Transfer in Mouse ...
1 Introduction
2 Materials
3 Methods
3.1 Identification of Adequate Donor-Recipient Pairs
3.1.1 Determining the MIC of Relevant Strains to Antibiotics Used in the Mouse Model
3.1.2 In Vitro Conjugation Tests Between Salmonella Donors and Enterobacteriaceae Recipients to Characterize Plasmids of Inter...
3.1.3 Identifying a Suitable Donor Strain in an Animal Model for Typhoidal Salmonellosis
3.1.4 Identifying a Suitable Recipient Strain in an Animal Model
3.1.5 Tagging of Plasmids with WITS Tags Coupled to Antibiotic Resistance Markers
3.2 Infection of Mice with the Donor and Antibiotic Treatment Schedule to Yield Tissue Lodged Persisters
3.2.1 Generation of Mixed WITS Inoculum
3.2.2 Animal Infection and Antibiotic Treatment Schedule
3.2.3 Euthanasia of Mice and Analysis of Persister Reservoirs and Population Sizes
3.2.4 Enrichment of S.Tm WITS from Fecal or Organ Homogenates
3.3 Introduce Recipient Strains by Oral Gavage and Analysis of Transconjugant Formation in the Gut Lumen
3.3.1 Infecting Mice Harboring Tissue-Lodged Persisters with Recipient Strains
3.3.2 Euthanasia of Mice at the End of the Experiment, Analysis of Conjugation Dynamics, and the Formation of Reservoirs of S....
3.4 Quantification of WITS Tag Abundance by qPCR
3.4.1 qPCR Analysis of WITS Tag Abundance
3.4.2 Analysis of the Population Structure and Detection of Bottlenecks or Rare Events
3.5 Modifications to the Protocol for Alternate Applications: Oral Infection with S.Tm Donor Strains as an Example
4 Notes
References
Chapter 18: Studying Antibiotic Persistence During Infection
1 Introduction
2 Materials
2.1 Quantification of Antibiotic Persisters In Vitro and In Vivo
2.2 Infection of Bone-Marrow Macrophages
2.3 Visualization of Heterogeneity in a Bacterial Population During Infection
2.4 Bacterial Persistence Using DualRNAseq
3 Methods
3.1 Studying Antibiotic Persistence In Vitro (Fig. 1a)
3.2 Studying Antibiotic Persistence In Vivo
3.2.1 Infection of Bone-Marrow Macrophages (BMM)
3.2.2 Quantification of Antibiotic Persisters In Vivo (Fig. 1b)
3.2.3 Tracking and Quantifying Growing and Non-growing Bacteria (Fig. 2)
3.2.4 Distinction Between Active and Inactive Non-growers (Fig. 3a)
3.2.5 Overview of Bacterial Persistence Using Dual RNAseq
4 Notes
References
Correction to: Studying Antibiotic Persistence During Infection
Index