Antibiotics: Methods and Protocols

Preface
Contents
Contributors
Part I: Production and Design
Chapter 1: Antibiotics: Precious Goods in Changing Times
1 The Times They Are Still Changing
2 Antibiotic Modes of Action
2.1 The Bacterial Cell Envelope as Antibiotic Target
2.2 Inhibitors of RNA and Protein Synthesis
2.3 Inhibitors of DNA Synthesis
2.4 Antibiotics with Extended or Novel Modes of Action
References
Chapter 2: A Whole-Cell Assay for Detection of Antibacterial Activity in Actinomycete Culture Supernatants
1 Introduction
2 Materials
2.1 Equipment
2.2 Media, Solvents, Culture Supernatants, and Reference Compound
2.3 Strains
3 Methods
3.1 Cultivation and Storage of B. subtilis ATCC 6051
3.2 Cultivation of Streptomyces fradiae DSM 41546 (Tylosin Production Assay)
3.3 Whole-Cell Assay for Detection of Antibacterial Activity
3.4 Data Evaluation Using an Example of a Whole-Cell Assay
3.5 Optional: HPLC Analysis of the Culture Supernatants of S. fradiae DSM 41546 (Tylosin Analytic)
4 Notes
References
Chapter 3: Sampling of Human Microbiomes to Screen for Antibiotic-Producing Commensals
1 Introduction
2 Materials
2.1 Consumables
2.2 Chemicals and Buffers
2.3 Media and Agar
2.4 Instruments and Equipment
2.5 Test Bacteria
2.6 Ethical Considerations
3 Methods
3.1 Collection of Microbiome Samples from the Nasal Cavities and Skin
3.2 Isolation of Potential Antimicrobial Producers (PAPs)
3.3 Storage of PAPs
3.4 Agar-Based Screening for Antimicrobial Activity
3.5 Soft Agar-Based Screening for Antimicrobial Activity
3.6 Liquid-Based Screening for Antimicrobial Activities
3.7 Species Identification of PAPs by MALDI-TOF MS
3.8 16s rDNA PCR-Based Species Identification
3.9 First Steps of Compound Identification
4 Notes
References
Chapter 4: Production of Antimicrobial Compounds by Homologous and Heterologous Expression
1 Introduction
2 Materials
2.1 Cloning Procedure
2.1.1 Instrumentation
2.1.2 Kits
2.1.3 Strains, Plasmids, and Primers
2.1.4 Chemicals
2.1.5 Others
2.2 Medium
2.3 Antibiotic and IPTG Stock Solutions
3 Methods
3.1 Cloning Procedure
3.1.1 Generating Electro-Competent Cells
3.1.2 Preparing the Ready-to-Use Gibson Assembly (GA) Master Mix
3.1.3 Transformation of E. coli by Electroporation
3.1.4 Generation of E. coli Cryo-Cultures for Storage
3.1.5 Generation of Streptomycete Spore Suspension for Storage
3.1.6 Restriction Analysis of Plasmid DNA
3.2 Construction of the Plasmid to Be Used for Conjugation and Homologous Recombination into the Genome of the Streptomyces Ho...
3.2.1 DNA Fragment of Homologous Regions 1 and 2
3.2.2 DNA Fragment of the Vector
3.2.3 DNA Fragment of the Apramycin Resistance Gene (Acc(3)IV) and oriT
3.2.4 Fusion of the Four DNA Fragments Using Gibson Assembly
3.2.5 Transfer of the Generated Plasmid to the Streptomyces Strain by Tri-Parental Conjugation
3.2.6 Cultivation of the Homologous Expression Host
3.3 Heterologous Expression of a BGC in Escherichia coli
3.3.1 Preparation of Gibson Assembly Fragments
3.3.2 Plasmid Construction by Gibson Assembly and Transferring the Heterologous Expression Construct into a Heterologous Host
3.3.3 Cultivation of Heterologous Expression Host
4 Notes
Appendix
Sequence S1
References
Chapter 5: Isolation and Purification of Natural Products from Microbial Cultures
1 Introduction
1.1 Product Isolation (Capture)
1.1.1 Capture from Biomass
1.1.2 Capture from Culture Filtrate
1.2 Product Purification
1.2.1 Principles of Chromatography
1.2.2 Chromatographic Techniques in Natural Product Recovery
1.3 Product Polishing
1.4 Practical Concerns in Natural Product Recovery
2 Materials
3 Methods
3.1 Development of a Recovery Strategy
3.1.1 Locate the Active Compound
3.1.2 Capture Active Compound from Biomass
3.1.3 Capture Active Compound from Culture Supernatant
3.1.4 Normal-Phase Chromatography in Pretest
3.2 Recovery of Fusarubin as an Example
3.2.1 Fungus Cultivation and Compound Capture
3.2.2 Fungal Extract Purification on Silica Column
3.2.3 Purification Fusarium-Containing Fractions on Size-Exclusion Column
3.2.4 Polishing Fusarubin on Preparative Reversed-Phase HPLC
4 Notes
References
Chapter 6: Structure Elucidation of Antibiotics
1 Introduction
2 Materials
2.1 Extract Analysis and Structure Dereplication by HPLC-(HR)MS
2.2 Structure Elucidation by NMR
3 Methods
3.1 Extract Analysis and Structure Dereplication by HPLC-(HR)MS
3.2 Sample Preparation for NMR Analysis
3.3 Useful Information from NMR Measurements
3.3.1 Chemical Shift
3.3.2 Coupling Constant
3.3.3 Integration
3.4 1D NMR Methods
3.4.1 1H NMR
3.4.2 13C NMR
3.5 2D NMR Methods
3.5.1 COSY
3.5.2 1H-13C- HMQC/HSQC
3.5.3 1H-13C HMBC
3.6 Partial Structure Elucidation of Tylosin A
4 Notes
References
Chapter 7: Computer-Aided Drug Design: An Update
1 Introduction
2 Materials
3 Methods
3.1 Protein Structure Prediction Using AlphaFold
3.2 MD Simulations with Polarizable Force Field
3.3 Docking Using SILCS-MC and ML-Based Reweighting for SAR
3.4 Binding Site Identification Using SILCS-Hotspots
3.5 Membrane Permeation Prediction Using SILCS
3.6 Protein-Protein Interaction Prediction Using SILCS-PPI
3.7 Biologic Formulation Using SILCS
4 Notes
References
Chapter 8: Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents
1 Introduction
2 Materials
2.1 Isolation of Human Neutrophils and Erythrocytes
2.2 LDH Activity
2.3 Cell Viability Assay Based on Resazurin
2.4 Cell Proliferation Assay Using WST-1
2.5 Cell Death Analysis with 7-Aminoactinomycin D (7-AAD)
2.6 Isolation and Cell Culture of Primary Human Keratinocytes
2.7 Cell Viability Assay Using 4-Methylumbelliferyl Heptanoate (MUH)
2.8 Hemolysis Assay
3 Methods
3.1 Isolation of Human Neutrophils and Erythrocytes
3.2 Isolation and Cultivation of Primary Human Keratinocytes
3.3 LDH Activity Assay
3.4 Cell Viability Assay Based on Resazurin
3.5 Cell Viability Assay Based on Cell Proliferation Reagent WST-1
3.6 Cell Death Analysis with 7-Aminoactinomycin-D (7-AAD)
3.7 Keratinocyte Cell Viability Analysis Using 4-Methylumbelliferyl Heptanoate (MUH)
3.8 Hemolysis Assay
4 Notes
References
Part II: Mode of Action and Resistance
Chapter 9: Microscopy-Based Multiwell Assay to Characterize Disturbed Bacterial Morphogenesis Upon Antibiotic Action
1 Introduction
2 Materials
2.1 Semiautomated, 96-Well-Based Microscopy Assay for Bacterial Phenotyping
2.1.1 Growth Media, Reagents/Materials, and Bacterial Strains
2.1.2 Hardware and Software
2.2 Phenotypic Profiling Using Fluorescence Microscopy
2.2.1 Growth Media, Reagents/Materials, and Bacterial Strains
2.2.2 Hardware and Software
3 Methods
3.1 Semiautomated, 96-Well-Based Microscopy Assay for Bacterial Phenotyping
3.1.1 Preparation of Bacterial Cultures
3.1.2 Preparation of 96-Well Plates for the Antibiotic Treatment of Bacteria
3.1.3 Preparation of Microplate-Sized Agarose Pads
3.1.4 Collection of Aliquots and Transfer onto Agarose Pads
3.1.5 Semiautomated Microscopy Workflow for Samples in a 96-Well Format
3.2 Phenotypic Profiling Using Fluorescence Microscopy
3.2.1 Preparation of Bacterial Cultures
3.2.2 Preparation of Agarose-Coated Microscopy Slides
3.2.3 Antibiotic Treatment of Bacterial Cells
3.2.4 Labeling of Samples with Fluorescent Dyes
3.2.5 Sample Preparation for Microscopy
3.2.6 Fluorescence Microscopy
4 Notes
References
Chapter 10: Expansion Microscopy of Bacillus subtilis
1 Introduction
2 Materials
2.1 Cultivation
2.2 Fixation and Staining
2.3 Expansion Microscopy
2.4 Sample Chamber, Image Acquisition, and Analysis
3 Methods
3.1 Cultivation
3.2 Fixation and Staining
3.3 Expansion Microscopy
3.4 Image Acquisition
4 Notes
References
Chapter 11: Tracking Global and Local Changes in Membrane Fluidity Through Fluorescence Spectroscopy and Microscopy
1 Introduction
2 Materials
2.1 Laurdan Fluorescence Spectroscopy In Vivo
2.2 Laurdan Fluorescence Spectroscopy In Vitro
2.3 Laurdan Fluorescence Microscopy
2.4 Nile Red Fluorescence Microscopy
2.5 DiIC12 Fluorescence Microscopy
3 Methods
3.1 Laurdan Fluorescence Spectroscopy In Vivo
3.1.1 Sample Preparation and Data Acquisition
3.1.2 Data Analysis
3.2 Laurdan Fluorescence Spectroscopy In Vitro
3.2.1 Lipid Handling
3.2.2 Vesicle Preparation
3.2.3 Fluorescent Spectroscopy Parameters
3.2.4 Data Acquisition
3.2.5 Data Analysis
3.3 Laurdan Fluorescence Microscopy
3.3.1 Sample Preparation
3.3.2 Microscope Calibration
3.3.3 Image Capture
3.3.4 Image Analysis
3.4 Nile Red Fluorescence Microscopy
3.5 DiIC12 Fluorescence Microscopy
3.6 B. subtilis Lipid-Modified Strains for the Analysis of Lipid Specificity of Antimicrobial Compounds
3.6.1 B. subtilis Strains with Tunable Phospholipid Composition
3.6.2 B. subtilis Strains with Tunable Fatty Acid Composition and Fluidity
3.6.3 Preparation of Precursors for Tunable Fatty Acid Composition Strain
4 Notes
References
Chapter 12: Quantitative Analysis of Microscopy Data to Evaluate Bacterial Responses to Antibiotic Treatment
1 Introduction
2 Materials
2.1 Growth Media, Reagents, and Bacterial Strains for (Time-Resolved) Fluorescence Microscopy
2.2 Hardware, Image Acquisition, and Analysis
2.3 Analysis Software and Plugins
3 Methods
3.1 Preparation of S. aureus or B. subtilis Cultures
3.2 Sample Preparation for Time-Lapse Experiments with S. aureus
3.3 Preparation of the Microscope for Time-Lapse Experiments
3.4 Microscopy of S. aureus in Time-Lapse Experiments
3.5 Deconvolution
3.6 Kymograph Analysis of S. aureus
3.7 Interpretation of Averaged Kymographs of S. aureus
3.8 Analysis of S. aureus Cells Using Convolved Average Projections (CAP)
3.8.1 Extraction of Individual Cell Images with MicrobeJ
3.8.2 Combine Images into a Convolved Average Projection
3.9 Analysis of Cell Length, Width, and Cell Count of Rod-Shaped B. subtilis, and Generation of Histograms Using Oufti Software
4 Notes
References
Chapter 13: Application of a Bacillus subtilis Whole-Cell Biosensor (PliaI-lux) for the Identification of Cell Wall Active Ant...
1 Introduction
2 Materials
2.1 Media and Reagents
2.2 Bacterial Strains
2.3 Special Equipment
3 Methods
3.1 ``Spot-on-Lawn´´ Reporter Screen
3.2 Luminescence Detection
3.3 Luminescence Quantification
4 Notes
References
Chapter 14: Determination of Bacterial Membrane Impairment by Antimicrobial Agents
1 Introduction
2 Material
2.1 Determination of Bacterial Membrane Potential Using [3H]tetraphenylphosphonium Bromide (TPP+)
2.1.1 Growth Medium, Bacterial Strains, and Antimicrobial Substances
2.1.2 Bacterial Membrane Potential Determination
2.1.3 Protein Determination of Whole Cells
2.2 Measurement of Potassium Release from Whole Cells
2.2.1 Growth Medium, Bacterial Strains, and Antimicrobial Substances
2.2.2 Potassium Efflux Measurement
3 Method
3.1 Determination of Bacterial Membrane Potential Using [3H]tetraphenylphosphonium Bromide (TPP+)
3.1.1 Measurement of TPP+ Uptake and Distribution
3.1.2 Protein Determination of Whole Cells
3.1.3 Calculation of Bacterial Membrane Potential
3.2 Measurement of Potassium Release from Whole Cells
3.2.1 Measurement of Potassium Efflux
3.2.2 Calculation of Released Potassium Concentration
4 Notes
References
Chapter 15: A Colorimetric Assay to Identify and Characterize Bacterial Primase Inhibitors
1 Introduction
2 Materials
2.1 Materials for Production and Purification of Enzymes
2.2 Equipment
2.3 Chemicals, Protease, and Buffer for Purification of Enzymes
2.4 Malachite Green Reagent Components
2.5 Stock Solutions, Buffers, and Solvents for Activity Assay
2.6 Activity Assay Reagents for Identification of DnaG Inhibitors
2.7 Materials for Activity Assay for Confirmation of PPiase Inhibitors
2.8 Materials for DNA Intercalation Assay
3 Methods
3.1 Overexpression of Protein in Escherichia coli (Ec) BL21 (DE3) Cells
3.2 Cell Lysis and Protein Purification
3.2.1 MtDnaG and BaDnaG
3.2.2 SaDnaG
3.2.3 MtPPiase and SaPPiase
3.2.4 EcPPiase
3.3 Activity Assay
3.3.1 Preparation of the Malachite Green Reagent
3.3.2 Testing of Assay Feasibility and Robustness
3.3.3 The High-Throughput Screening Assay (384-Well Plate)
3.3.4 Medium-Throughput Screening Assay (96-Well Plate)
3.3.5 Assessment of Results
3.4 Bacterial PPiase Inhibition
3.5 The Dose-Response Assay and Inhibitor Selectivity
3.6 Mode of Inhibition
3.7 Identification of DNA Binding Compounds by DNA Intercalation Assay
4 Notes
References
Chapter 16: Cell-Based Fluorescent Screen Amenable to HTS to Identify Inhibitors of Bacterial Translation Initiation
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Culture Media
2.3 Chemicals and Equipment
3 Methods
3.1 Outline of the Assay Cascade
3.2 Fluorescent Assay Protocol
4 Notes
References
Chapter 17: Bacterial Two Component Systems: Overexpression and Purification: In Vitro and In Vivo Inhibitor Screens
1 Introduction
2 Materials
2.1 Expression and Purification of a Recombinant Full-Length Histidine Kinase in Escherichia coli
2.2 Phosphorylation Assay Using Radioactively Labeled ATP
2.3 Phosphorylation Assay Using Phos-tag Acrylamide
2.4 Construction of WalR-FLAG Tagged Strains
2.5 In Vivo Phosphorylation Assay Using Phos-tag Acrylamide and Western Blotting
3 Methods
3.1 Recombinant Expression and Purification of a Full-Length Histidine Kinase in Escherichia coli (see Note 15)
3.2 Phosphorylation Assay Using Radioactively Labeled ATP
3.3 Phosphorylation Assay Using Phos-tag Acrylamide
3.4 Construction of the WalR-FLAG Strain
3.5 In Vivo Phosphorylation Assay Using Phos-tag Acrylamide and Western Blotting
4 Notes
References
Chapter 18: Sample Preparation for Mass Spectrometry-Based Absolute Quantification of Bacterial Proteins in Antibiotic Stress ...
1 Introduction
2 Materials
2.1 Cell Counting
2.2 Cell Harvest and Lysis
2.3 Determination of Protein Concentration
2.4 Enrichment of Membrane Proteins
2.5 In-Solution Digest of Cytosolic Proteins and Sample Purification
2.6 S-Trap Digest of Membrane Proteins and Sample Purification
3 Methods
3.1 Cell Counting
3.2 Cell Harvest and Lysis
3.3 Determination of Protein Concentration
3.4 Enrichment of Membrane Proteins
3.5 In-Solution Digest of Cytosolic Proteins and Sample Purification
3.6 S-Trap Digest of Membrane Proteins and Sample Purification
4 Notes
References
Chapter 19: Elemental Analysis for the Characterization of Antimicrobial Effects
1 Introduction
2 Materials
2.1 Cultivation of B. subtilis in Belitzky Minimal Medium (See Notes 1 and 2)
2.2 Sample Preparation
2.3 Sample Measurement and Data Evaluation
3 Methods
3.1 Cultivation of B. subtilis
3.2 Sample Preparation (See Note 23)
3.3 Sample Measurement and Data Evaluation
4 Notes
References
Chapter 20: Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research
1 Introduction
2 Materials
2.1 Cultivation of B. subtilis in Belitzky Minimal Medium (BMM) (See Note 1)
2.2 Ribosome Isolation by Ultracentrifugation
2.3 Tryptic Digest of Isolated Ribosomes
2.4 UPLC-MSE Analysis
2.5 Label-Free Quantitation of MSE Data
3 Methods
3.1 Cultivation of B. subtilis for Ribosome Isolation (See Note 13)
3.2 Cell Disruption and Ribosome Isolation (See Note 21)
3.3 Tryptic Digest (See Note 28)
3.4 Mass Spectrometry
3.5 Label-Free Quantitation of Proteins
3.6 Manual Analysis of Regulated Proteins (See Note 32)
4 Notes
References
Chapter 21: Functional Metagenomics to Study Antibiotic Resistance
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 Construction of Metagenomic Library
2.3 Nextera Library Preparation and Sequencing
2.4 Antibiotics and Media
2.5 Instruments and Glassware
2.6 Primers
3 Methods
3.1 Metagenomic DNA Extraction (see Note 11)
3.2 Metagenomic Library Preparation
3.2.1 Plasmid Preparation
3.2.2 Insert Preparation
3.2.3 Ligation and Dialysis
3.2.4 Electroporation, Metagenomic Library Amplification, and Quantification
3.3 Screening for Antibiotic Resistance and Amplification of Antibiotic Resistance-Conferring DNA Fragments
3.4 Illumina Library Preparation and Sequencing
3.5 Assembly and Annotation of ARGs
3.5.1 Demultiplexing and Preprocessing of Sequencing Reads
3.5.2 De Novo Assembly of Short-Read Sequences Using PARFuMS
3.5.3 Annotation of Contigs with Antibiotic Resistance Functions
4 Notes
References
Index