Antifungal Drug Resistance: Methods and Protocols

Preface
Contents
Contributors
Part I: Identification and Characterization of Molecules with Antifungal Activity
Chapter 1: Antifungal Susceptibility of Yeasts and Filamentous Fungi by CLSI Broth Microdilution Testing
1 Introduction
2 Materials
2.1 Growth Media, Chemicals, and Disposables
2.2 Equipment
3 Methods
3.1 RPMI Growth Medium (see Note 1)
3.2 Preparation of Antifungal Top Antifungal Stock Solution
3.3 Preparation of Drug Sets at 100x Concentration Range
3.4 Microtiter Plate Preparation
3.5 Inoculum Preparation: Yeast
3.6 Inoculum Preparation: Molds (see Note 4)
3.7 Inoculation of Plates
3.8 Incubation of Plates and Endpoint Reading
3.9 Interpretation of Results
4 Notes
References
Chapter 2: Luciferase-Based High-Throughput Screen with Aspergillus fumigatus to Identify Antifungal Small Molecules
1 Introduction
2 Materials
2.1 Glucose Minimal Medium (GMM) for A. fumigatus Culture
3 Methods
3.1 Growth/Propagation/Conidia Preparation for High-Throughput Screen
3.2 High-Throughput Screening Assays
4 Basic Protocols
4.1 Preparation of Glucose Minimal Medium (GMM)
4.2 Preparation of Conidia
4.3 Making Freezer Stocks of Conidia Preparations
4.4 HTS Screening Protocol
4.5 Viability Measurements
4.6 Calculations (See Note 6)
4.7 Data Management (See Note 7)
4.8 Hit Confirmation Assays and Counter-Screens
4.9 Dose-Response Assays
5 Example Screen
5.1 Materials
5.2 Optimized Assay Protocol
5.3 Results
6 Notes
References
Chapter 3: A Dual-Readout High-Throughput Screening Assay for Small Molecules Active Against Aspergillus Fumigatus
1 Introduction
2 Materials
2.1 Preparation of Conidia
2.2 Preparation of Z´ Plate and Plate Inoculation
2.3 Development of AK
3 Methods
3.1 Preparation of Conidia
3.2 Preparation and Inoculation of Z´ Plate Using Automated Plate Filler
3.3 Development of AK Assay
3.4 Data Analysis
4 Notes
References
Chapter 4: Antifungal Susceptibility Testing and Drug Discovery in the Dimorphic Fungus Histoplasma Capsulatum
1 Introduction
2 Materials
2.1 Preparation of Histoplasma Yeast Cells
2.2 Titer Histoplasma Cell Suspension
2.3 Microdilution Assay
2.4 Intramacrophage Susceptibility Testing
3 Methods
3.1 Preparation of Histoplasma Yeast Cells
3.2 Titer Histoplasma Cell Suspension
3.3 Microdilution Assay
3.4 Intramacrophage Antifungal Susceptibility Testing
4 Notes
References
Chapter 5: A 384-Well Microtiter Plate Model for Candida Biofilm Formation and Its Application to High-Throughput Screening
1 Introduction
2 Materials
2.1 Fungal Strains
2.2 Microbiological Media
2.3 Phosphate-Buffered Saline (PBS)
2.4 XTT
2.5 Menadione
2.6 Antifungal Drugs
2.7 Sterile Disposable Plasticware and Glassware
2.8 384-Well Microtiter Plates
2.9 Compound Libraries
2.10 Hematocytometer
2.11 Microtiter Plate Reader
2.12 Biosafety Cabinet
2.13 Automated Liquid Handling
3 Methods
4 Notes
References
Part II: Identification and Characterization of Fungal Strains with Altered Drug Susceptibility
Chapter 6: Protocols for Measuring Tolerant and Heteroresistant Drug Responses of Pathogenic Yeasts
1 Introduction
2 Materials
2.1 Culture Preparation
2.2 Broth Microdilution Assay
2.3 Disk Diffusion Assay
2.4 Population Analysis Profiling
2.5 ColonyScanalyser
3 Methods
3.1 Culture Conditions
3.2 Broth Microdilution Assays
3.3 Disk Diffusion Assays
3.4 Population Analysis Profiling (PAP) Assay
3.5 ColonyScanalyser
4 Notes
References
Chapter 7: Genome-Wide Identification of Variants Associated with Antifungal Drug Resistance
1 Introduction
2 Materials
2.1 Sequence Data
2.2 Phenotypic Data
2.3 Software
3 Methods
3.1 Setup
3.2 Quality Control and Evaluation of Sequence Data
3.3 Formatting the Reference Genome
3.4 Prepare Input Files
3.5 Read Alignment, Variant Identification (SNP/Indel), and Filtering
3.6 Variant Annotation
3.7 Quality Control Checks for Variant Calls
3.8 Copy Number Variation (CNV) Analysis
3.9 Comparison of Serial or Clonally Related Isolates
3.10 Genotype-Phenotype Association
4 Notes
References
Chapter 8: Copy Number Variation and Allele Ratio Analysis in Candida albicans Using Whole Genome Sequencing Data
1 Introduction
2 Materials
3 Methods
3.1 Visualization of Whole Genome Sequencing Read Depth Using Yeast Mapping Analysis Pipeline (YMAP)
3.2 Alignment of FASTQ Files to a Reference Genome
3.3 Use Samtools to Generate Raw Read Depth Values
3.4 Use RStudio to Identify CNVs and Determine CNV Breakpoints in Whole Genome Sequencing Data
3.5 Identification of Heterozygous Allele Frequencies for CNV Detection
3.6 Visualize Allele Frequencies
4 Notes
References
Chapter 9: Drug Target Elucidation Through Isolation and Analysis of Drug-Resistant Mutants in Cryptococcus neoformans
1 Introduction
2 Materials
2.1 Growth Media and Reagents
2.2 Consumables
2.3 Equipment
3 Methods
3.1 Culture Growth and Preparation
3.2 Solid Media Selection of Drug-Resistant Spontaneous Mutants
3.3 Drug Diffusion Selection of Drug-Resistant Spontaneous Mutants
3.4 Drug Diffusion of Known Antifungal Drugs from ETEST Strips
3.5 Liquid Passaging and Evolution of Resistance in the Presence of Drug
3.6 Maintenance of Drug-Resistant Mutants
3.7 Determination of Drug-Resistant Mutant Stability
3.8 Broth Microdilution to Determine Minimum Inhibitory Concentration (MIC)
3.9 Genomic DNA Extraction for Illumina Whole-Genome Sequencing (WGS) to Determine Causative Mutation(s) in Spontaneous Drug-R...
3.10 Identification of Variants in Drug-Resistant Mutants
3.11 Genetic Crossing of Drug-Resistant Strains to Wild-Type Reference Strains to Segregate SNPs and Resistance Phenotype (Cry...
4 Notes
References
Chapter 10: Chemical-Genetic Approaches for Exploring Mode of Action of Antifungal Compounds in the Fungal Pathogen Candida al...
1 Introduction
2 Materials
3 Methods
3.1 Preparing Pooled Aliquots of the C. albicans DBC
3.2 Preparing Compound-Treated Cultures
3.3 Genomic DNA Isolation
3.4 Genomic DNA Quantification
3.5 Barcode PCR
3.6 Barcode Sequencing Library Creation and Purification
3.7 Data Analysis
4 Notes
References
Part III: Characterization of the Genetic and Molecular Mechanisms of Antifungal Resistance
Chapter 11: Characterizing Candida glabrata Pdr1, a Hyperactive Transcription Factor Involved in Azole Resistance
1 Introduction
2 Materials
2.1 Cloning of PDR1-GOF-natMX6-cre-EBD Evictable System into pUC19
2.2 Insertion and Characterization of PDR1-GOF Alleles in Genome of C. glabrata
3 Methods
3.1 Cloning of PDR1-GOF-natMX6-cre-EBD Evictable System into pUC19
3.2 Insertion and Characterization of PDR1-GOF Alleles in Genome of C. glabrata
4 Notes
References
Chapter 12: Heterologous Expression of Candida Antifungal Target Genes in the Model Organism Saccharomyces cerevisiae
1 Introduction
2 Materials
3 Methods
3.1 Amplification of Gene of Interest and Digestion of Chosen Plasmid
3.2 Co-Transformation of Competent S. cerevisiae Cells with PCR and Digested Plasmid
3.3 Colony PCR Screen of Transformants and Confirmation of Gene Sequence
4 Notes
References
Chapter 13: Conditional Protein Depletion in the Analysis of Antifungal Drug Resistance in Candida glabrata
1 Introduction
2 Materials
2.1 PCR Amplification and Purification of ScLEU2-OsTIR1 and 3XminiAID-5XFLAG-HIS3MX6 Cassettes
2.2 Integration of ScLEU2-OsTIR1 and 3XminiAID-5XFLAG-HIS3MX6 Cassettes at Desired Loci in the C. glabrata Genome
2.3 Analysis of the Effect of Acute Snf2 Depletion on Fluconazole-Induced Gene Expression
3 Methods
3.1 PCR Amplification and Purification of ScLEU2-OsTIR1 and 3XminiAID-5XFLAG-HIS3MX6 Cassettes
3.2 Integration of the ScLEU2-OsTIR1 and 3XminiAID-5XFLAG-HIS3MX6 Cassettes in the Genome of C. glabrata
3.3 Analysis of the Effect of Acute Snf2 Depletion on Fluconazole-Induced Gene Expression
3.4 Significance
4 Notes
References
Chapter 14: Transport across Membranes: Techniques for Measuring Efflux in Fungal Cells
1 Introduction
2 Materials
3 Methods
3.1 Rhodamine Assay in Yeast
3.2 Ala-Nap Accumulation in Yeast
3.3 Ala-Nap Accumulation in Filamentous Fungi
4 Notes
References
Chapter 15: Transport Across Membranes: Techniques for Measuring Drug Import in Fungal Cells
1 Introduction
2 Materials
3 Methods
3.1 Yeast Cultures
3.2 Filamentous Fungal Cultures
4 Notes
References
Part IV: Characterization of Antifungal Drug Resistance Using Animal Infection Models
Chapter 16: Analysis of Candida Antifungal Resistance Using Animal Infection Models
1 Introduction
2 Materials
2.1 Animals
2.1.1 Rat Biofilm Model
2.1.2 Mouse Disseminated Model
2.2 Medications
2.2.1 Rat Biofilm Model
2.2.2 Mouse Disseminated Model
2.3 Surgical and Infection Materials
2.3.1 Rat Biofilm Model
2.3.2 Mouse Disseminated Model
2.4 Media
2.5 Evaluation of Selected Endpoints
2.5.1 Microbiologic Counts
2.5.2 Confocal or Fluorescent Microscopy of Catheter Biofilms (Optional)
2.5.3 Scanning Electron Microscopy of Catheter Biofilms (Optional)
3 Methods
3.1 Rat Biofilm Model (see Note 1)
3.1.1 Preparation of Catheters
3.1.2 Preparation of Surgical Equipment
3.1.3 Catheter Placement
3.1.4 Animal and Catheter Maintenance
3.1.5 Preparation of Inoculum
3.1.6 Infection of Catheter
3.1.7 Antifungal Lock Treatment of Catheter
3.1.8 Harvesting the Catheter
3.1.9 Endpoint Microbiological Counts
3.1.10 Endpoint Fluorescent or Confocal Microscopy (Optional)
3.1.11 Endpoint Scanning Electron Microscopy (Optional)
3.2 Mouse Disseminated Model
3.2.1 Animal Preparation and Maintenance
3.2.2 Preparation of Inoculum
3.2.3 Disseminated Tail Vein Infection
3.2.4 Antifungal Treatment
3.2.5 Harvesting the Kidneys
3.2.6 Endpoint Determination Microbiological Counts
3.2.7 Data Interpretation
4 Notes
4.1 Rat Biofilm Model
4.2 Mouse Disseminated Model
References
Chapter 17: Using Bioluminescence to Image Candida glabrata Urinary Tract Infections in Mice
1 Introduction
2 Materials
2.1 Preparation of the Inoculum
2.2 Infection
2.3 Measurement
2.4 Quantification
3 Methods
3.1 Preparation of the Inoculum
3.2 Infection
3.3 Monitoring of the Animals
3.4 Bioluminescence Imaging of the Infection
3.5 Colony-Forming Unit Quantification of Infection
4 Notes
References
Index