Antibiotic Resistance Protocols (Methods in Molecular Biology, 2833)

Preface
	References
Contents
Contributors
Chapter 1: Using the Zebrafish Larval Model of Infection to Investigate Antibiotic Efficacy and Combination Treatments Against...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Choice of Dug Concentrations Based on In Vitro Minimum Inhibitory Concentration
		3.2 Toxicity Assays and Choice of Solvent
		3.3 Antibiotic Stability Assay
		3.4 Titration of Antibiotic Doses (Survival Curves)-Infected  Fish
		3.5 Determination of Live Bacterial Numbers With/Without Antibiotics
	4 Notes
	References
Chapter 2: Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension
	1 Introduction
	2 Materials
		2.1 Culture Media
		2.2 RCCS Culture System
	3 Methods
		3.1 Preparation of Media
		3.2 RCCS Inoculum Preparation
		3.3 Inoculation of the RCCS Culture System
		3.4 Establishing and Maintaining Cell Aggregates in the RCCS System
		3.5 Harvesting Biofilms from the  RCCS
	4 Notes
	References
Chapter 3: Rapid Gene Silencing Followed by Antimicrobial Susceptibility Testing for Target Validation in Antibiotic Discovery
	1 Introduction
	2 Materials
		2.1 Golden Gate Assembly
		2.2 Colony PCR
		2.3 Agarose Gel Electrophoresis
		2.4 Plasmid DNA Extraction and Sequencing
		2.5 Transformation into Mycobacterium abscessus
		2.6 Validation of Gene Silencing
		2.7 Gene Essentiality Testing Using HT-SPOTi
	3 Methods
		3.1 CRISPRi Mutant Generation Using Golden Gate Assembly
		3.2 Agarose Gel Electrophoresis
		3.3 Plasmid DNA Extraction and Sequencing
		3.4 Transformation into M. abscessus
		3.5 Validation of Gene Silencing
		3.6 HT-SPOTi: Investigating Gene Essentiality
	4 Notes
	References
Chapter 4: Modified HT-SPOTi: An Antimicrobial Susceptibility Testing to Evaluate Formulated Therapeutic Combinations Against ...
	1 Introduction
	2 Materials
		2.1 Growth Medium
		2.2 Drug Testing
		2.3 In Vitro Modified Spot Growth Inhibition Assay
	3 Methods
		3.1 Preparation of Bacterial and Mycobacterial Cultures
		3.2 Preparation of Master Plate for Drug Testing
		3.3 Agar Plate Preparation
		3.4 Adding Drugs to Prepared Agar Plate
		3.5 Spotting Bacterial Cultures
	4 Notes
	References
Chapter 5: Investigating Combination Therapy as a Means to Enhance Activity and Repurpose Antimicrobials
	1 Introduction
		1.1 Definition and Types of Combination Therapy
		1.2 Synergy
		1.3 Definition of Synergy
	2 Materials
		2.1 Bacterial Growth
	3 Methods
		3.1 Broth Microdilution (BMD)
		3.2 Checkerboard
	4 Notes
	References
Chapter 6: Investigating Photoactive Antimicrobials as Alternatives (or Adjuncts) to Traditional Therapy
	1 Introduction
	2 Materials
		2.1 Growth of Microorganisms
		2.2 aPDT
	3 Methods
		3.1 Bacterial Strains and Growth Conditions
		3.2 Photosensitizer Solution
		3.3 Toxicity of Methylene Blue in the   Dark
		3.4 Photodynamic Therapy Tests
	4 Notes
	References
Chapter 7: Using Hollow Fiber to Model Treatment of Antimicrobial-Resistant Organisms
	1 Introduction
		1.1 Understanding Hollow Fiber Bioreactors
		1.2 Advantages of Using Hollow Fiber Bioreactors
		1.3 Applications of Hollow Fiber Bioreactors in Antimicrobial Resistance Studies
		1.4 Limitations and Considerations
		1.5 Initial Conclusions
	2 Materials
	3 Methods
		3.1 Hollow Fiber Bioreactor
		3.2 Sampling
		3.3 Sample Examination
	4 Notes
	References
Chapter 8: A Microtiter Plate Assay at Acidic pH to Identify Potentiators that Enhance Pyrazinamide Activity Against Mycobacte...
	1 Introduction
	2 Materials
		2.1 Growth Medium
		2.2 Viability Detection Reagents
		2.3 Additional Reagents
		2.4 Equipment
	3 Methods
		3.1 Preparation of M. tuberculosis Inoculum
		3.2 Growth of M. tuberculosis in Middlebrook 7H9 for 5 Days
		3.3 Growth of M. tuberculosis in Acidified Middlebrook 7H9 for 7 Days
		3.4 Growth of M. tuberculosis in Acidified Middlebrook 7H9 for 10 Days (Microtiter Plates) in the Presence of Antibiotic Combi...
			3.4.1 Preparation of PZA-RIF and PZA-NOR Combinations in 96-Well Plates (Chequerboard)
			3.4.2 Inoculation of Antibiotic Dose Matrix Microtiter Plates with M. tuberculosis
		3.5 Endpoint Detection/Final Viability Assessment
		3.6 Data Analysis
	4 Notes
	References
Chapter 9: Within-Host Mathematical Models of Antibiotic Resistance
	1 Introduction
	2 Within-Host Modeling
	3 Types of Models: Compartmental Models and IBMs
	4 Future Developments: More Data Is Needed
	References
Chapter 10: Use of Individual-Based Mathematical Modelling to Understand More About Antibiotic Resistance Within-Host
	1 Introduction
	2 Constructing IBMs
		2.1 Defining the Environment and the Individual Elements
		2.2 Declaration of Initial State Variables (and Traits) of the Individual Entities
		2.3 Defining Individual Activities/Actions and Environmental Attributes Influencing State (and Traits) Updates
		2.4 Declaration of Assumptions About Behavioral Rules of Individuals
		2.5 Defining the Initialization of State (and Trait Variables)
		2.6 Defining Parameter Input
		2.7 Decision on Result Display
	References
Chapter 11: Monitoring Live Mycobacteria in Real-Time Using a Microfluidic Acoustic-Raman Platform
	1 Introduction
	2 Materials
		2.1 Bacteria, Culture Conditions, and Contamination Control
		2.2 Microfluidic Chamber
		2.3 Raman Spectroscopy System
		2.4 An Environmental Control Enclosure
		2.5 Precision Syringe  Pump
	3 Methods
		3.1 Determining MIC
		3.2 Optimization of Experimental Parameters
		3.3 Raman Spectroscopy of Trapped Bacteria
	4 Notes
	References
Chapter 12: Phylogenetic Survival Analysis
	1 Introduction
	2 Materials
		2.1 Computing
		2.2 Software
	3 Methods
		3.1 Pre-processing
		3.2 Genome Alignment
		3.3 Phylogenetic Inference
		3.4 Ancestral State Reconstruction
		3.5 Ancestral Sequence Reconstruction
		3.6 Phylogenetic Survival Analysis
		3.7 Output
	4 Notes
	References
Chapter 13: Rapid Drug Susceptibility Testing to Preserve Antibiotics
	1 Introduction
	2 What Do We Mean by Fast?
	3 Speeding the Laboratory Process
	4 Indirect Detection of Bacterial Growth by Detecting Metabolic Change
		4.1 The BD Phoenix Automated Microbiology System
		4.2 Vitek 2
		4.3 Microscan
	5 Direct Bacterial Detection
		5.1 Detecting Metabolic Components
		5.2 Detecting Growth Through Bacterial Scattering
		5.3 Optical Morphological Reading Methods
		5.4 Camera Systems
		5.5 Atomic Force Microscopy
	6 Detecting Volatile Compounds (Electronic Nose)
	7 Direct Detection by Molecular Means
	8 Whole Genome Sequencing for Drug Susceptibility Testing
	9 Summary and Conclusion
	References
Chapter 14: Quantifying Viable M. tuberculosis Safely Obviating the Need for High Containment Facilities
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Materials
	3 Methods
		3.1 Sputum Specimens
			3.1.1 Heat Inactivation (see Note 4)
			3.1.2 GTC Inactivation (see Note 4)
		3.2 RNA Extraction
		3.3 DNA Removal
		3.4 Preparation of Master   Mix
		3.5 Thermal Cycling Conditions
	4 Notes
	References
Chapter 15: Improved Diagnosis and Treatment Monitoring of Tuberculosis Using Stool and the Tuberculosis Bacterial Load Assay ...
	1 Introduction
	2 Materials
		2.1 Materials for Stool Collection
		2.2 General Laboratory Requirements
		2.3 M. tuberculosis RNA Extraction
	3 Methods
		3.1 Collecting the Stool Sample
		3.2 Processing Stool Samples
		3.3 Preparation of Probes and Primers Notes for the TB-MBLA Study
		3.4 RNA Extraction
		3.5 DNAse Treatment
		3.6 Preparation of RT-qPCR Master Mix (Use QuantiTec Multiplex RT-PCR NR Kit (QT))
		3.7 Reverse Transcriptase   qPCR
	4 Notes
	References
Chapter 16: Application of Pathogen Genomics to Outbreak Investigation
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 DNA Quality Assessment and Quantification
		2.3 Library Preparation
		2.4 MiSeq Sequencer Preparation
		2.5 Illumina Sequencing
	3 Methods
		3.1 DNA Extraction
		3.2 DNA Quality Assessment and Quantification
			3.2.1 DNA Purity Check on a NanoDrop Spectrophotometer
			3.2.2 DNA Molecular Weight and Fragmentation Assessment by Agarose Gel Electrophoresis
			3.2.3 DNA Quantity
			3.2.4 Sample and Sequencing Kit Selection
		3.3 Library Preparation
			3.3.1 Tagmentation (see Note 6)
			3.3.2 Neutralization
			3.3.3 PCR Amplification (see Note 7)
			3.3.4 Post-PCR Clean-Up
			3.3.5 Manual Library Normalization and Pooling (see Note 11)
		3.4 Preparing PhiX Control (see Note 15)
		3.5 Preparing the MiSeq Instrument
		3.6 Denaturing the Pooled Library
		3.7 Beginning the Sequencing Run
	4 Notes
	References
Chapter 17: Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing
	1 Introduction
	2 Materials
		2.1 DNA-Library Preparation Adapted from Baym et al.
		2.2 DNA Sequencing
	3 Methods
		3.1 DNA-Library Preparation Adapted from Baym et al.
		3.2 DNA Sequencing on the NextSeq 500 Platform
		3.3 Run Evaluation of a NextSeq 500  Run
	4 Notes
	References
Chapter 18: Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing: From S...
	1 Introduction
		1.1 Overview Bioinformatics Analysis of MTBC NGS Data
	2 Methods
		2.1 Identifying Drug Resistance Determinants with MTBseq
		2.2 Generating Resistance Reports
		2.3 Comparative Genomic Analysis
		2.4 Alternative Tools for MTBC Antimicrobial Resistance Prediction
		2.5 Tool for the Prediction of Antibiotic Resistances in Non-tuberculous Mycobacteria (NTMs)
	3 Notes
	References
Chapter 19: Analysis of Whole Genome Sequencing Data for Detection of Antimicrobial Resistance Determinants
	1 Introduction
	2 Materials
	3 Methods
		3.1 Getting Started
		3.2 Identification and Typing of Data
			3.2.1 KmerFinder-Prediction of Bacterial Species Using a Fast K-Mer Algorithm
			3.2.2 MLST-Multi Locus Sequence Typing (MLST) from an Assembled Genome or from a Set of Reads
		3.3 Identification of Antimicrobial Resistance Determinants
			3.3.1 ResFinder-Detection of Acquired and Point Mutations Associated with Antimicrobial Resistance
	4 Notes
	References
Index