Mycobacterium ulcerans: Methods and Protocols

Preface
Contents
Contributors
Part I: Mycobacterium ulcerans Disease and Host Pathogen Interaction
	Chapter 1: Overview: Mycobacterium ulcerans Disease (Buruli Ulcer)
		1 Introduction
		References
	Chapter 2: Immunohistochemistry: A Method to Analyze M. ulcerans Infected Skin Tissue
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 3: Primary Isolation of Mycobacterium ulcerans
		1 Introduction
		2 Materials
			2.1 Pathological Specimens, Decontamination, and Culture
			2.2 Environmental Sample Processing, Decontamination, and Culture
		3 Methods
			3.1 Processing of Pathological Specimens
			3.2 Decontamination of Pathological Specimens and Culture
			3.3 Purification and Identification of Bacterial Colonies
			3.4 Processing of Environmental Samples
			3.5 Decontamination of Environmental Samples and Culture
			3.6 Purification and Identification of Bacterial Colonies
		4 Notes
		References
	Chapter 4: Investigation of Mycobacterium ulcerans Glycan Interactions Using Glycan Array and Surface Plasmon Resonance
		1 Introduction
		2 Materials
			2.1 Glycan Array Analysis of Whole Cells
			2.2 SPR Analysis of Whole Cells Using Amine Coupling (C1) Sensor Chips (BIAcore)
			2.3 Glycan Array Analysis of His-Tagged Recombinant Proteins
			2.4 SPR Analysis of Recombinant Proteins Using Amine Coupling (CM5) Sensor Chips (BIAcore)
		3 Methods
			3.1 Glycan Array Analysis of Whole Cells
			3.2 SPR Using Bacteria Cells Immobilized onto a C1 Sensor Chip
			3.3 Glycan Array Analysis of Tagged Recombinant Proteins
			3.4 SPR Analysis of Recombinant Proteins Using Amine Coupling (CM5) Sensor Chips (BIAcore)
		4 Notes
		References
	Chapter 5: Purification and Characterization of Extracellular Vesicles from Mycobacterium ulcerans Culture
		1 Introduction
		2 Materials
			2.1 Purification of Extracellular Vesicles from M. ulcerans Culture
			2.2 Quantification of Purified Vesicles
			2.3 Quantification of Mycolactone from Purified Vesicles
			2.4 Cellular Tests
		3 Methods
			3.1 Purification of Extracellular Vesicles from M. ulcerans Culture
			3.2 Quantification of Purified Vesicles
			3.3 HPLC Quantification of Mycolactone from Purified Vesicles
			3.4 Cellular Tests
		4 Notes
		References
	Chapter 6: DNA Extraction from Clinical Specimens for the Direct Detection of Mycobacterium ulcerans by Real-Time PCR
		1 Introduction
		2 Materials
			2.1 Extraction of DNA from Swabs and Fresh Tissue
			2.2 Extraction of DNA from Paraffin-Embedded Formalin-Fixed Tissue Sections
		3 Methods
			3.1 Pre-extraction Specimen Preparation for Swabs and Fresh Tissue
			3.2 Pre-extraction Specimen Preparation for Paraffin-Embedded Formalin-Fixed Tissue Sections (See Note 8)
			3.3 Lysis of Mycobacteria in Fresh Specimens Using Alkali and Heat
			3.4 DNA Cleanup from Crude Lysates
			3.5 Lysis of Mycobacteria in Fixed Tissue Specimens
			3.6 DNA Cleanup Following Proteinase K Digestion of Fixed Tissue Samples
		4 Notes
		References
	Chapter 7: Extraction of DNA from Environmental Samples for the Real-Time PCR Detection of Mycobacterium ulcerans
		1 Introduction
		2 Materials
			2.1 Extraction of DNA from Environmental Samples
		3 Methods
			3.1 Extraction of DNA from Liquid Samples Including Water, Biofilms, and Sediment
			3.2 Extraction of DNA from Soil, Detritus, and Feces
			3.3 Extraction of DNA from Vegetation Samples
			3.4 Extraction of DNA from Insects
		4 Notes
		References
	Chapter 8: Detection of Mycobacterium ulcerans DNA Using Real-Time PCR
		1 Introduction
		2 Materials
			2.1 Detection of M. ulcerans DNA by Real-Time PCR
				2.1.1 Mastermix Preparation (Perform in Dedicated PCR Mastermix Room) (See Notes 1-3)
				2.1.2 Addition of DNA Template (Perform in Dedicated PCR Template Room) (See Notes 1 and 3)
				2.1.3 Amplification and Detection of PCR Products
		3 Methods
			3.1 Detection of M. ulcerans DNA by Real-Time PCR
				3.1.1 Preparation of TaqMan Mastermix (Perform in Dedicated PCR Mastermix Room)
				3.1.2 Adding DNA Extracts and Controls to the Mastermix (Perform in Dedicated PCR Template Room)
				3.1.3 Amplification and Detection of PCR Products
				3.1.4 Interpretation of Results
		4 Notes
		References
	Chapter 9: Determining Viability of M. ulcerans by 16S rRNA RT Reverse Transcriptase Real-Time PCR
		1 Introduction
		2 Materials
			2.1 Sample Collection
			2.2 Combined M. ulcerans RNA/DNA extraction and Purification
			2.3 Reverse Transcription
			2.4 Combined M. Ulcerans Viability Assay
		3 Methods
			3.1 Simultaneous RNA/DNA Extraction
			3.2 RNA Purification
			3.3 Reverse Transcription of Total RNA to cDNA
			3.4 Whole Genome DNA Purification
			3.5 PCR
		4 Notes
		References
	Chapter 10: Methods and Approaches for Buruli Ulcer Surveillance in Africa: Lessons Learnt and Future Directions
		1 Introduction
		2 Considerations of Health System Structure
		3 Passive Surveillance, Clinical Diagnosis, and Data Reporting
		4 Community Outreach, Mobilization, and Stigma Reduction
		5 Active Case Finding and the Role of Community Health Workers
		6 Survey-Based Approaches and Burden Estimation
		7 Improved Targeting and Evaluation of Surveillance
		8 Laboratory Confirmation in Surveillance Programs
		9 Integration of BU with Other Skin-NTDs
		10 Conclusion
		References
Part II: Purification and Quantification of Myolactone
	Chapter 11: Overview: Mycolactone, the Macrolide Toxin of Mycobacterium ulcerans
		1 Introduction
		References
	Chapter 12: Conditions for Handling and Optimal Storage of Mycolactone
		1 Introduction
		2 Materials
		3 Methods
			3.1 Storage
			3.2 Sample Collection
			3.3 Handling
			3.4 Decontamination
		4 Notes
		References
	Chapter 13: Mycolactone Purification from M. ulcerans Cultures and HPLC-Based Approaches for Mycolactone Quantification in Bio...
		1 Introduction
		2 Materials
			2.1 Mycolactone Extraction from M. ulcerans Cultures
			2.2 Mycolactone Extraction from Biological Samples
			2.3 Mycolactone Analysis by LC-MS/MS
		3 Methods
			3.1 Extraction of Mycolactone from Bacterial Cultures
			3.2 Extraction of Mycolactone from Tissues
			3.3 Extraction of Mycolactone A/B from Biological Fluids
			3.4 LC-MS/MS Detection
				3.4.1 Chromatographic Separation
				3.4.2 Mass Spectrometry Method
		4 Notes
		References
	Chapter 14: Detection of Mycolactone by Thin Layer Chromatography
		1 Introduction
		2 Materials
			2.1 Optimized TLC Method
			2.2 Semi-Quantification of Mycolactone on TLC
			2.3 Preparation of 0.1 M Solution of 2-Naphthylboronic  Acid
			2.4 Sampling Techniques
				2.4.1 Fine Needle Aspiration
				2.4.2 Swab Specimen
				2.4.3 Biopsy
			2.5 Sample Extraction
				2.5.1 Method for TLC Analysis of Tissue Samples
				2.5.2 Preparation of 10 ng/μL Mycolactone A/B
				2.5.3 Mouse Foot Pad Studies
			2.6 TLC Analysis of Urine Samples
				2.6.1 M. ulcerans-Infected BALB/c  Mice
				2.6.2 Mycolactone-Spiked Urine Samples
				2.6.3 Urine Samples from BU Patients
		3 Methods
			3.1 Optimized TLC Method
			3.2 Semi-Quantification of Mycolactone by TLC
			3.3 Sampling Techniques
				3.3.1 Fine Needle Aspiration (FNA)
				3.3.2 Swab Specimen
				3.3.3 Biopsies
			3.4 TLC Analysis of Biological Samples
				3.4.1 Clinical Samples from BU Patients (Fig. 5)
				3.4.2 Mouse Footpad Samples
			3.5 TLC Analysis of Urine Samples
				3.5.1 Urine from M. ulcerans-Infected BALB/c  Mice
				3.5.2 TLC Analysis of Spiked Urine Samples (Fig. 6)
				3.5.3 TLC Analysis of Patients´ Urine
		4 Notes
		References
	Chapter 15: Competitive ELISA for the Detection and Quantification of Mycobacterium ulcerans Mycolactone
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 ELISA
			2.3 Antigens and Conjugates
		3 Methods
			3.1 Sample Preparation
			3.2 ELISA
			3.3 Analysis
		4 Notes
		References
	Chapter 16: Biochemical and Biological Assays of Mycolactone-Mediated Inhibition of Sec61
		1 Introduction
		2 Materials
			2.1 Mycolactone Solutions
			2.2 Biochemical Assays
				2.2.1 In Vitro Transcription
				2.2.2 In Vitro Translation
			2.3 Biological Assays
				2.3.1 Cell Culture and Activation
				2.3.2 Flow Cytometry Analysis
				2.3.3 ELISA
		3 Methods
			3.1 Biochemical Assays
				3.1.1 In Vitro Transcription
				3.1.2 In Vitro Translation
			3.2 Biological Assays
				3.2.1 Downregulation of CD62L Surface Expression
				3.2.2 Inhibition of Cytokine Production
		4 Notes
		References
Part III: Drug Development Against Mycobacterium ulcerans
	Chapter 17: Overview: Development of Drugs Against Mycobacterium ulcerans
		1 Introduction
		References
	Chapter 18: In Vitro Assessment of Drug Activity Against Mycobacterium ulcerans
		1 Introduction
		2 Materials
			2.1 Preparation of Bacterial Inoculum
			2.2 Preparation of Drug-Containing Media
		3 Methods
			3.1 Preparation of Middlebrook 7H9 Media
			3.2 Preparation of Bacterial Inoculum
			3.3 Preparation of Middlebrook 7H11 Media
			3.4 Determination of MIC with Agar Proportion Method
		4 Notes
		References
	Chapter 19: Drug Efficacy Testing in the Mouse Footpad Model of Buruli Ulcer
		1 Introduction
		2 Materials
		3 Methods
			3.1 Inoculum Preparation
			3.2 Footpad Inoculation
			3.3 Assessment of Footpad Swelling
			3.4 Footpad Harvest
			3.5 Drug Treatment
			3.6 Assessment of Treatment Response
			3.7 Determination of Drug Efficacy in  Mice
		4 Notes
		References
	Chapter 20: Turbidity-Based MIC Assay and Characterization of Spontaneous Drug Resistant Mutants in Mycobacterium ulcerans
		1 Introduction
		2 Materials
			2.1 Middlebrook 7H9 Medium
			2.2 M. ulcerans Frozen Stocks
			2.3 Middlebrook 7H11 Agar in Quad-Plates
			2.4 Middlebrook 7H11 Agar Plates with Antibiotics
			2.5 Determination of MIC in Broth
			2.6 Inoculation of 7H11 Plates
			2.7 Genomic DNA Extraction for Targeted Sequencing
		3 Methods
			3.1 Revival and Growth of Stock Strain in 7H9 Liquid Medium
			3.2 Determination of MIC Using a Direct Microbroth MIC Assay
			3.3 Inoculation of 7H11 Plates to Raise Resistant Mutants
			3.4 Accurate Determination of the Inoculation  Size
			3.5 Subculture of Mutant Colonies and Propagation in Liquid Medium
			3.6 Genomic DNA Extraction and Sequencing
			3.7 Calculation of Mutation  Rate
		4 Notes
		References
	Chapter 21: Determination of Bioenergetic Parameters in Mycobacterium ulcerans
		1 Introduction
		2 Materials
			2.1 Middlebrook 7H9 Medium
			2.2 M. ulcerans Frozen Stocks and Revival/Growth
			2.3 Test Drug Stocks
			2.4 Measurement of  ATP
			2.5 Measurement of OCR (MitoXpress Assay)
			2.6 Measurement of OCR with the Seahorse XF Analyzer
		3 Methods
			3.1 Revival and Growth of Stock Strain in 7H9 Liquid Medium
			3.2 Preparation of Drugs (Stock and Intermediate Concentration) for ATP Depletion Assay
			3.3 Measurement of ATP Depletion with BacTiter-Glo Reagent
			3.4 Measurement of OCR with the MitoXpress Xtra Oxygen Consumption Assay
			3.5 Measurement of OCR with the Seahorse XFe96 Analyzer
		4 Notes
		References
Index