Preface
Contents
Contributors
Part I: Isolation and Identification of Candida auris
Chapter 1: Isolation of Candida auris in Clinical Specimens
1 Introduction
2 Collection, Transportation, and Processing of Samples for Isolation of Candida auris from Clinical Specimens
2.1 Blood Culture
2.2 Sterile Body Fluids (Cerebrospinal, Peritoneal, Synovial, Vitreous Humor, etc.)
2.3 Respiratory Specimens (Sputum, Bronchial Aspirate, Bronchoalveolar Lavage)
2.4 Urine
2.5 Abscess (Exudates/Pus/Wound Material) and Tissue Specimens)
2.6 Surveillance Cultures from Body Sites for Detection of Colonization by Candida auris
3 Phenotypic Characteristics of Candida auris
4 Isolation of Candida auris from Clinical Samples Harboring Mixed Yeast Flora
5 Selective Enrichment Broth for Isolation of Candida auris from Surveillance Cultures
6 Identification of Candida auris by Commercial Biochemical-Based Systems
7 MALDI-TOF Mass Spectrometry-Based Identification of Candida auris
8 Molecular-Based Identification Methods for Candida auris
8.1 Sequence-Based Identification of Candida auris Isolates
8.2 Direct Detection of Candida auris DNA in Clinical Specimens
8.3 Nucleic Acid-Based Amplification Testing Candida auris from Yeast Colonies
8.3.1 Method: PCR and Real-Time qPCR (SYBR Green)
8.3.2 Method: Duplex PCR
8.3.3 Method: Tetraplex PCR
8.3.4 Method: Multiplex PCR
8.3.5 Method: YEAST PANEL Multiplex PCR
8.3.6 Method: GPS MONODOSE dtec-qPCR kit
8.4 Nucleic Acid Amplification Testing of Candida auris for Surveillance Studies
8.4.1 Method: Real-Time PCR (TaqMan)
8.4.2 Method: Real-Time PCR (SYBR Green)
8.4.3 Method: T2 Magnetic Resonance (T2MR) System
8.4.4 Method: Loop-Mediated Isothermal Amplification (LAMP)
9 Conclusion
References
Chapter 2: Candida auris Identification and Profiling by MALDI-ToF Mass Spectrometry
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Basic Consumables and Chemicals
3 Methods
3.1 Preanalytical Procedures
3.2 Sample Preparation Procedures
3.2.1 Preparation and Handling of Organic Solvent
3.2.2 Preparation and Handling of Matrix
3.2.3 Preparation and Handling of 70% Formic Acid
3.2.4 Full Extraction Protocol (FX)
3.2.5 Partial Extraction Protocol (PX)
3.2.6 On-Target Plate Extraction Protocol (OTL)
3.2.7 Running the Target Plate on the MALDI-ToF MS Platform
3.2.8 Result Interpretation
3.2.9 Candida auris Mass Spectra Profiling
4 Notes
References
Chapter 3: Molecular Tools for Candida auris Identification and Typing
1 Introduction
2 Materials
2.1 Polymerase Chain Reaction
2.2 Gel Electrophoresis
2.3 Sequencing (Sanger Method)
2.4 Fragment Analysis
3 Methods
3.1 Internal Transcribed Spacer (ITS) Sequence-Based Identification
3.1.1 Polymerase Chain Reaction
3.1.2 Gel Electrophoresis
3.1.3 Sequencing
3.2 Microsatellite Typing
3.2.1 Polymerase Chain Reaction
3.2.2 Gel Electrophoresis
3.2.3 Fragment Analysis
4 Notes
References
Chapter 4: qPCR Detection of Candida auris Using the GPS CanAur MONODOSE dtec-qPCR Test
1 Introduction
2 Materials
2.1 DNA Extraction Using the GPStraction DNA Colonies Kit
2.2 GPS CanAur MONODOSE dtec-qPCR Test for C. auris Detection
3 Methods
3.1 GPStraction DNA Colonies Kit
3.2 qPCR for C. auris Detection
3.2.1 Preparation of Standard Curve Dilution Series
3.2.2 qPCR
3.2.3 Recommended Reaction Controls
3.2.4 Interpretation of Results (see Note 13)
3.2.5 Validation Methods
4 Notes
References
Part II: Testing Susceptibility of Candida auris to Antifungal Drugs
Chapter 5: Susceptibility Testing of Candida auris Isolates
1 Introduction
2 Materials
2.1 Culture Media
2.2 Buffers
2.3 Antifungal Agents
2.4 Solvents
2.5 Suspension Fluid
2.6 Equipment
2.7 Fungal Isolates
3 Methods
3.1 Preparation of SAC Media and Culturing of Yeast Isolates
3.2 MIC Testing
3.2.1 Preparation of RPMI Medium
3.2.2 Preparation of Drug Dilutions (see Note 7)
3.2.3 Preparation of Plates for the Microdilution Test
3.2.4 Preparation of Inoculum
3.2.5 Inoculation and Reading of Microtiter Plates and Interpreting the Results
3.3 Gradient Strip Methodology for Assessing Antifungal Susceptibility of Candida auris Isolates (see Note 13)
3.3.1 Inoculum Preparation
3.3.2 Setting Up the Gradient Strip Test
3.3.3 Interpretation of Gradient Strip Test Results
4 Notes
References
Chapter 6: Deciphering Candida auris Paradoxical Growth Effect (Eagle Effect) in Response to Echinocandins
1 Introduction
2 Materials
2.1 Fungal Culture
2.2 Broth Microdilution
2.3 DNA Extraction
2.4 Amplification of Hotspot 1 (HS1) of FKS1 Gene of C. auris
2.5 Gel Electrophoresis
2.6 Sanger Sequencing
2.7 FKS1 HS1 Sequence Analysis
3 Methods
3.1 Antifungal Susceptibility Testing
3.2 FKS1 HS1 Sequence Analysis
4 Notes
References
Part III: Genetic Manipulation of Candida auris
Chapter 7: An Optimized Electroporation Procedure for Genetic Transformation of Candida auris
1 Introduction
2 Material
3 Methods
3.1 Preparation of the Transforming DNA
3.2 Preparation of C. auris Electro-Competent Cells
3.3 Electroporation of C. auris Cells
4 Notes
References
Chapter 8: Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol
1 Introduction
2 Materials
2.1 General Materials
2.2 Fusion PCR
2.3 Transformation
2.4 Checking Transformants
2.5 Genomic DNA Extraction
3 Methods
3.1 Obtaining the Transformation Cassette by Fusion PCR
3.2 C. auris Transformation
3.3 Transformant Screening
3.4 Extraction of High-Quality Genomic DNA (gDNA)
4 Notes
References
Chapter 9: Downregulation of Essential Genes in the Fungal Pathogen Candida auris
1 Introduction
2 Materials
3 Methods
3.1 Generation of Gene-Specific Promoter Replacement DNA Cassette
3.2 C. auris Transformation
3.3 Genotyping Transformants
3.4 Confirm Doxycycline-Repressible Expression
3.5 Confirm Essentiality of Gene of Interest
4 Notes
References
Part IV: Phenotypic and Biochemical Characterization of Candida auris
Chapter 10: Cell Viability Assays for Candida auris
1 Introduction
2 Materials
2.1 Candida auris Storage, Revival, and Cell Standardization
2.2 Metabolic Assays and Biomass Assessment
2.3 Viable Cell Quantification
2.4 Microscopy Imaging
3 Methods
3.1 Cell Standardization
3.2 Planktonic and Biofilm Preparation
3.3 Metabolic Activity Assessment Using Microtiter Plate Assays
3.3.1 XTT Assay
3.3.2 Resazurin Assay
3.4 Cell Counting and Live/Dead Quantitative PCR
3.4.1 Biofilm Preparation and Sonication for Viable Counts
3.4.2 Cell Counting Using the Miles and Misra Technique
3.4.3 Viable and Total Cell Count Quantification Using Live/Dead qPCR
3.5 Live/Dead Fluorescent Imaging
4 Notes
References
Chapter 11: Measuring Gene Expression via mRNA Abundance in Candida auris
1 Introduction
2 Materials
2.1 Growth
2.2 RNA Isolation and cDNA Preparation
2.3 RT-qPCR
3 Methods
3.1 Cell Growth
3.2 RNA Isolation and Quantification
3.3 cDNA Synthesis
3.4 RT-qPCR and Expression Analysis
3.4.1 Primer Design and Optimization
3.4.2 RT-qPCR Reaction Setup and Selection of Endogenous Controls
3.4.3 Expression Profile Analysis
4 Notes
References
Chapter 12: Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Candida auris Metabolites
1 Introduction
2 Material
3 Methods
3.1 Sample Preparation
3.2 Sample Extraction
3.3 Derivatization
3.4 GC-MS
3.5 Data Analysis
3.6 Data Interpretation and Software
3.6.1 Excel
3.6.2 MetaboAnalyst
3.6.3 R-package
3.7 Statistical Analysis
4 Notes
References
Chapter 13: Isolation of Extracellular Vesicles from Candida auris
1 Introduction
2 Materials
3 Methods
3.1 Establishing the Cultures and Pre-inoculum Preparation
3.1.1 EV Isolation from Liquid Media
3.1.2 EV Isolation from Solid Media
3.2 EV Sedimentation Following Collection of EVs from Either Liquid or Solid Media
3.3 Quantification and Visualization of EVs
4 Notes
References
Chapter 14: Labelling of Candida auris Cell Walls to Examine Levels of PAMP Exposure by Flow Cytometry and Fluorescence Micros...
1 Introduction
2 Materials
3 Methods
3.1 Labelling Cells
3.2 Microscopy
3.3 Flow Cytometry
4 Notes
References
Chapter 15: High-Pressure Freezing and Transmission Electron Microscopy to Visualize the Ultrastructure of the C. auris Cell W...
1 Introduction
2 Materials
2.1 Culturing of C. auris
2.2 High-Pressure Freezing (HPF)
2.3 Freeze Substitution (FS)
2.4 Ultrathin Sectioning and Staining of TEM Grids
3 Methods
3.1 Preparation of C. auris Cells
3.2 HPF
3.3 Freeze Substitution (FS)
3.4 Sectioning of TEM Samples
3.5 Staining of TEM Samples
4 Notes
References
Part V: Bioinformatic Methods for Candida auris
Chapter 16: Generating Complete Genome Assemblies of Candida auris
1 Introduction
2 Materials
2.1 Sequence Data
2.2 Software
3 Methods
3.1 Computational Environment
3.2 Quality Control and Evaluation of Sequence Data
3.3 Long-Read Assembly
3.4 Genome Structure Evaluation and Manual Finishing
3.5 Long-Read Polishing
3.6 Short-Read Polishing
3.7 Quality Control Checks
4 Notes
References
Chapter 17: MycoSNP: A Portable Workflow for Performing Whole-Genome Sequencing Analysis of Candida auris
1 Introduction
2 WGS Analysis
2.1 Selecting a Reference Genome
2.2 Repeat Identification and Masking
2.3 Indexing Reference Genome
2.4 Downsampling Reads
2.5 Quality Control of Reads
2.6 Read Alignment
2.7 Variant Calling
2.8 Generating a FASTA File
3 MycoSNP Workflow Implementation
3.1 BWA Reference Workflow
3.2 BWA Pre-processing
3.3 GATK Variants
3.4 Installation of MycoSNP
4 Conclusion
5 Notes
References
Chapter 18: Bioinformatic Identification of ABC Transporters in Candida auris
1 Introduction
2 Materials
2.1 Data Files
2.2 Computational Resources
3 Methods
3.1 Prediction of Potential ABC Proteins Based on NBD-HMM
3.2 Identification of Putative Transporter Proteins Within the ABC Repertoire of C. auris
4 Conclusions
5 Notes
References
Part VI: Exploring Host-Pathogen Interactions of Candida auris
Chapter 19: Examining Neutrophil-Candida auris Interactions with Human Neutrophils Ex Vivo
1 Introduction
2 Materials
2.1 Candida auris Propagation
2.2 Neutrophil Isolation
2.3 Fungal Killing Assay
2.4 Phagocytosis Assay
2.5 ROS Assay
3 Methods
3.1 Organisms and Inoculum
3.2 Human Neutrophil Collection
3.3 Killing Assay
3.4 Phagocytosis Assay
3.5 Measurement of ROS
4 Notes
References
Chapter 20: Modeling Candida auris Colonization on Porcine Skin Ex Vivo
1 Introduction
2 Materials
2.1 Harvesting and Cleansing of the Pig Skin
2.2 Obtaining Ex Vivo Samples by Biopsy
2.3 Candida auris Propagation
2.4 Assessment of C. auris Colonization on Porcine Skin Samples
3 Methods
3.1 Harvesting and Cleansing of the Pig Skin
3.2 Obtaining Ex Vivo Samples by Biopsy
3.3 Organisms and Inoculum
3.4 Assessment of C. auris Colonization on Porcine Skin Samples
3.5 Viable Burden
3.6 Histopathology
4 Notes
References
Chapter 21: A Human Ex Vivo Skin Model to Study Candida auris Biofilms
1 Introduction
2 Materials
2.1 Fungal Strains and Growth Media
2.2 Equipment
2.3 Ex Vivo Human Skin Models to Study Fungal Infection
2.4 Recovery and Long-Term Storage of Samples
2.5 Scanning Electron Microscopy (SEM)
3 Methods
3.1 Inoculum Preparation
3.2 Preparation and Infection of Skin Explants
3.3 Recovery and Long-Term Storage of Samples
3.4 Confirmation of Biofilm Formation on Catheters and Yeast Growth on Skin Explants by SEM
4 Notes
References
Chapter 22: In Vivo Skin Colonization and Decolonization Models for Candida auris
1 Introduction
2 Materials
2.1 C. auris Inoculum Preparation
2.2 Guinea Pig Immunosuppression and Inoculation
2.3 Guinea Pig Treatments
2.4 Guinea Pig Euthanasia and Sample Collection
2.5 Colonization of the Mouse Skin
2.6 Mouse Decolonization Treatment
2.7 Mouse Euthanasia and Sample Collection
3 Methods
3.1 Candida auris Cutaneous Infection Guinea Pig Model
3.1.1 Inoculum Preparation
3.1.2 Immunosuppression and Inoculation
3.1.3 Treatments
3.1.4 Clinical Evaluation
3.1.5 Euthanasia and Mycological Evaluation
3.1.6 Histological Evaluation
3.2 Candida auris Skin Decolonization Mouse Model
3.2.1 Colonization of the Skin
3.2.2 Decolonization Treatment and Clinical Evaluation
3.2.3 Euthanasia and Mycological Evaluation
3.2.4 Histological Evaluation
4 Notes
References
Chapter 23: The Use of Galleria mellonella Larvae to Study the Pathogenicity and Clonal Lineage-Specific Behaviors of the Emer...
1 Introduction
2 Materials
3 Methods
3.1 Harvesting of Fungal Cultures and Preparation of Yeast Inocula
3.2 Inoculation of Galleria Larvae
3.3 Analysis of C. auris Pathogenicity in Inoculated Larvae
3.3.1 Using Larval Survival/Death as Primary Endpoint
3.3.2 Additional Health Endpoints: Activity, Melanization, Cocoon Formation, and the Larval Health Index Scoring System
3.3.3 Fungal Burden in Infected Larvae
3.3.4 Measuring Hemocyte Density (see Note 9)
3.4 Physical Examination of Fungal Cell Morphology in Infected Larvae
3.5 Evaluation of Antifungal Efficacy in Galleria mellonella
4 Notes
References
Chapter 24: Drosophila melanogaster as a Rapid and Reliable In Vivo Infection Model to Study the Emerging Yeast Pathogen Candi...
1 Introduction
2 Materials
2.1 Housing and Manipulation of Flies
2.2 Culture of C. auris Isolates and Inoculum Preparation
2.3 Determination of Fungal Burden
2.4 General Equipment and Consumables
3 Methods
3.1 Preparation of Antifungal Drug-Supplemented Fly Food Vials
3.2 Antifungal (Pre)treatment of Flies
3.3 Preparation of the C. auris Inoculum Suspension
3.4 Infection of Flies with C. auris Yeast Cells
3.5 Post-Infection Monitoring
3.6 Determination of Fungal Burden
4 Notes
References
Chapter 25: An Immunocompromised Mouse Model of Candida auris Systemic Infection
1 Introduction
2 Materials
2.1 C. auris Inoculum Preparation
2.2 Mice Immunosuppression and Inoculation
2.3 Treatment Administration
2.4 Euthanasia and Sample Collection
2.5 Survival Analysis and Determination of Tissue Fungal Burden
3 Methods
3.1 Inoculum Preparation
3.2 Mice Immunosuppression, Inoculation, and Treatment Administration
3.3 Euthanasia and Sample Collection
3.4 Survival Analysis and Determination of Tissue Fungal Burden
3.4.1 Survival Analysis
3.4.2 Determination of Tissue Fungal Burden
4 Notes
References
Chapter 26: Mouse Gastrointestinal Colonization Model for Candida auris
1 Introduction
2 Materials
3 Methods
3.1 Inoculum Preparation
3.2 Mouse Model
3.3 End of Experiment: Culling and Dissecting
3.4 Stool and Organ CFU Determination (See Note 9)
3.5 Statistical Analysis
3.6 Reporting Animal Experiments
4 Notes
References
Index