Listeria Monocytogenes: Methods and Protocols 2nd Edition

Preface
Contents
Contributors
Part I: Detection, Quantification, and Confirmation
	Chapter 1: Traditional Methods of Analysis for Listeria monocytogenes
		1 Introduction
		2 Materials
			2.1 Selective Enrichment Broth Media
			2.2 Isolation Selective Media
			2.3 Chromogenic Media
			2.4 Nonselective Media
		3 Methods
			3.1 Detection of L. monocytogenes
			3.2 Enumeration of L. monocytogenes
			3.3 Most Probable Number (MPN) of L. monocytogenes
			3.4 Confirmation
		4 Notes
		References
	Chapter 2: MALDI-ToF MS: A Rapid Methodology for Identifying and Subtyping Listeria monocytogenes
		1 Introduction
		2 Materials
			2.1 Laboratory Equipment
			2.2 Basic Consumables and Chemicals
			2.3 Software
		3 Methods
			3.1 Pre-analytical Procedures
			3.2 Sample Preparation Procedure
			3.3 Identification of Listeria monocytogenes Using MALDI-ToF  MS
			3.4 Generating a Consensus Spectra for Subtyping Using MALDI-ToF  MS
			3.5 Chemometric Analysis of Consensus Data for Subtyping Listeria monocytogenes
		4 Notes
		References
	Chapter 3: Sample Preparation for qPCR Detection of Listeria from Food
		1 Introduction
		2 Materials
			2.1 Basic Consumables and Buffers
			2.2 Lysis Buffers and Application Fields
			2.3 Duplex qPCR Assay for Quantification of L. monocytogenes and  ISPC
		3 Methods
			3.1 Preparation and Application of the Internal Sample Process Control (ISPC)
			3.2 Matrix Lysis
			3.3 Matrix Lysis Support Protocol for Meat and Fish Samples
			3.4 DNA Extraction
			3.5 DNA Standard Preparation for qPCR Quantification of L. monocytogenes and L. monocytogenes Delta-prfA/+IAC
		4 Notes
		References
	Chapter 4: qPCR Validation on the Basis of the Listeria monocytogenes prfA Assay
		1 Introduction
		2 Materials
			2.1 Media
			2.2 qPCR-Assay for Quantification of L. monocytogenes Detecting prfA
		3 Methods
			3.1 Standard Methods for qPCR Assay Performance Validation Parameters
				3.1.1 Calibration Curve
				3.1.2 Agarose  Gel
			3.2 Boundary Limit Analysis for qPCR Validation
				3.2.1 Limiting Dilution Assay
				3.2.2 Quantitative Poisson Analysis
			3.3 PCR-Stop Analysis
		4 Notes
		References
Part II: Subtyping Approaches
	Chapter 5: Serotype Assignment by Sero-agglutination, ELISA, and PCR
		1 Introduction
		2 Materials
			2.1 Agglutination Method
			2.2 ELISA Method
			2.3 PCR Method
		3 Methods
			3.1 Agglutination Method
			3.2 ELISA Method
			3.3 PCR Method
		4 Notes
		References
	Chapter 6: Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Listeria monocytogenes
		1 Introduction
		2 Materials
			2.1 Plating for Confluent Growth and Preparing Plugs
			2.2 Restriction Digestion of Plugs
			2.3 Casting the  Gel
			2.4 Electrophoresis and Staining
		3 Methods
			3.1 Preparation of Plugs from Agar Cultures
			3.2 Cell Lysis in Agarose Plugs
			3.3 Washing of Agarose Plugs After Cell Lysis
			3.4 Restriction Digestion of DNA in Agarose Plugs
			3.5 Casting Agarose Gel
			3.6 Loading Restricted Plugs into the Wells
			3.7 Electrophoresis Conditions
			3.8 Staining and Documentation of PFGE Agarose Gel
		4 Notes
		References
	Chapter 7: Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) of Listeria monocytogenes and Listeria innocua
		1 Introduction
			1.1 Seven Housekeeping Gene-Based MLST Scheme
			1.2 Whole Genome Sequencing-Core Genome-Based MLST
			1.3 The Idea Behind Core Genome-Based MLST (cgMLST)
		2 Materials
			2.1 Cultivation and Storage of L. monocytogenes Isolates
			2.2 DNA Extraction
			2.3 PCR Amplification
			2.4 DNA Purification for PCR Products
			2.5 Quality Control of Specific PCR Amplicons by Agarose Gel Electrophoresis
		3 Methods
			3.1 Storage and Cultivation of L. monocytogenes Isolates
			3.2 DNA Extraction
			3.3 PCR Amplification
			3.4 DNA Purification
			3.5 Quality Control of Specific PCR Amplicons by Agarose Gel Electrophoresis
			3.6 Sequencing and Cluster Analysis
		4 Whole Genome Sequencing and Data Analysis
			4.1 Cultivation and Genomic DNA Isolation
			4.2 Whole Genome Sequencing
		5 Notes
		References
Part III: Genotypic and Phenotypic Characterization
	Chapter 8: High-Throughput Characterization of Listeria monocytogenes Using the OmniLog Phenotypic Microarray
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Cell Suspensions
			3.2 Inoculation and Incubation of PM Plates
		4 Notes
		References
	Chapter 9: High-Throughput Screening of Biofilm Formation of Listeria monocytogenes on Stainless Steel Coupons Using a 96-Well...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Cleaning Solutions
			3.2 Preparation of Media and Diluent Solution
			3.3 Prepare Overnight Cultures of L. monocytogenes Isolates
			3.4 Preparation of Microplates
			3.5 Processing of Coupons
		4 Notes
		References
	Chapter 10: Confocal Laser Microscopy Analysis of Listeria monocytogenes Biofilms and Spatially Organized Communities
		1 Introduction
		2 Materials
			2.1 Bacterial Strains
			2.2 Labware and Reagents
			2.3 Equipment
		3 Methods
			3.1 L. monocytogenes Biofilms and Microcolony Preparation
				3.1.1 Bacterial Cultures
				3.1.2 Biofilm Formation on the Flat Bottom of Polystyrene Microscopic Grade 96-Well Microplates
				3.1.3 Biofilm Formation on Glass Microspheres
				3.1.4 Biofilm Formation in Multichannel Flow Cells
				3.1.5 Macrocolony on  Agar
				3.1.6 Microcolonies in (on) Axenic Soft Cheese Model
					Preparation of Miniature Axenic Soft Cheeses
			3.2 Noninvasive Confocal Laser Microscopy (CLM) Analysis of L. monocytogenes 3D Communities
				3.2.1 CLM Image Acquisitions Under the Microscope
				3.2.2 CLM Image Analysis
		4 Notes
		References
	Chapter 11: Extraction and Preparation of Listeria monocytogenes Subproteomes for Mass Spectrometry Analysis
		1 Introduction
		2 Materials
			2.1 Recovering All Subfractions by Cell Fractionation Approach
			2.2 Proteosurfaceome-Targeted Approaches
				2.2.1 Biotinylation of Cell Surface Proteins
				2.2.2 Trypsin Shaving of Cell Surface Proteins
			2.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
				2.3.1 SDS-PAGE
				2.3.2 Protein Staining and Band Treatment After Excision
		3 Methods
			3.1 Cell Fractionation Approach
				3.1.1 Cell Wall Proteins and Exoproteins
				3.1.2 Intracellular Proteins
				3.1.3 Membrane Proteins
			3.2 Biotinylation Approach
				3.2.1 Biotin Labeling
				3.2.2 Purification of Biotinylated Proteins
			3.3 Shaving Approach
			3.4 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
				3.4.1 SDS-PAGE
				3.4.2 Protein Staining and Band Treatment After Excision
		4 Notes
		References
Part IV: Strain Manipulation
	Chapter 12: Extraction and Analysis of Plasmid DNA from Listeria monocytogenes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Bacterial Cultures for Plasmid Isolation
			3.2 Isolation of Plasmids from Listeria monocytogenes Strains
			3.3 Assessment of Quality of Isolated Plasmid DNA from Listeria monocytogenes
		4 Notes
		References
	Chapter 13: Generation of Nonpolar Deletion Mutants in Listeria monocytogenes Using the ``SOEing´´ Method
		1 Introduction
		2 Materials
			2.1 SOE-PCR
			2.2 Cloning of SoeAD into Vector pKSV7
			2.3 Competent L. monocytogenes for Transformation
			2.4 Transformation of pKSV7(SoeAD) into L. monocytogenes Via Electroporation
			2.5 Chromosomal Integration of SoeAD
		3 Methods
			3.1 SOE-PCR
				3.1.1 Primer Design (See Note 2)
				3.1.2 SoeAB-SoeCD PCR Reaction
				3.1.3 SoeAD PCR Reaction
			3.2 Cloning of SoeAD into Vector pKSV7
			3.3 Competent L. monocytogenes for Transformation
			3.4 Transformation of pKSV7(SoeAD) into L. monocytogenes via Electroporation
			3.5 Chromosomal Integration of SoeAD Using pKSV7 (Fig. 2)
		4 Notes
		References
	Chapter 14: Mutant Construction and Integration Vector-Mediated Genetic Complementation in Listeria monocytogenes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Purification of Plasmid pMC38 Carrying a Mariner-Based Transposition System (TC1/Mariner)
			3.2 Preparation of Electrocompetent L. monocytogenes and Electroporation
			3.3 Preparation of E. coli Electrocompetent Cells and Electroporation
			3.4 Real-Time PCR
			3.5 Determination of Transposon Insertion Sites
			3.6 Genetic Complementation
		4 Notes
		References
Part V: Host-Pathogen Interactions
	Chapter 15: Internalization Assays for Listeria monocytogenes
		1 Introduction
		2 Materials
			2.1 Gentamicin Protection Assay
			2.2 Differential Bacterial Staining Assay
		3 Methods
			3.1 Gentamicin Protection Assay
			3.2 Differential Bacterial Staining
		4 Notes
		References
	Chapter 16: Microscopy of Intracellular Listeria monocytogenes in Epithelial Cells
		1 Introduction
		2 Materials
			2.1 Long-Term Infection Immunofluorescence Assay
			2.2 Immunofluorescence Assay to Study the Recruitment of Cytoskeletal Proteins During Infection
			2.3 LIVE/DEAD Viability Assay of Intracellular Listeria
		3 Methods
			3.1 Long-Term Infection Immunofluorescence Assay
			3.2 Immunofluorescence Assay to Study the Recruitment of Cytoskeletal Proteins During Infection
			3.3 LIVE/DEAD Viability Assay of Intracellular Listeria
		4 Notes
		References
Part VI: Control Methods
	Chapter 17: Control of Listeria monocytogenes Biofilms in a Simulated Food-Processing Environment
		1 Introduction
		2 Materials
			2.1 Estimation of Biofilm-Forming Ability by Microtiter Plate Assay
			2.2 Estimation of Biofilm-Forming Ability by Biofilm Cells Enumeration
			2.3 Evaluation of Biofilm Formation with Fluorescence Microscopy
			2.4 Surface Disinfection
			2.5 Biofilm Eradication Concentration
			2.6 Competitive Bacterial Species
			2.7 Bacteriophages
		3 Methods
			3.1 Biofilm Formation and Estimation-Microtiter Plate Assay
			3.2 Estimation of Biofilm-Forming Ability on Inert Surfaces by Cell Enumeration
			3.3 Evaluation of Biofilm Formation with Fluorescence Microscopy
			3.4 Surface Disinfection
			3.5 Biofilm-Eradicating Concentration
			3.6 Competitive Bacterial Species
			3.7 Bacteriophages
		4 Notes
		References
	Chapter 18: Sampling the Food-Processing Environment: Taking Up the Cudgel for Preventive Quality Management in Food Processin...
		1 Introduction
		2 Why to Use FPE Samples as Sampling Matrix?
		3 The Sensitivity of the Method Decides
		4 Concluding Remarks
		References
	Chapter 19: Isolation and Evaluation of Anti-Listeria Lactococcus lactis from Vegetal Sources
		1 Introduction
		2 Materials
			2.1 Isolation of LAB from Vegetal Sources
			2.2 Screening for Anti-Listeria Activity of LAB
			2.3 Identification of Anti-Listeria LAB and Bacteriocin Genes
		3 Methods
			3.1 Isolation of LAB from Vegetal Sources
			3.2 Screening for Anti-Listeria Activity of LAB
			3.3 Identification of Anti-Listeria LAB and Bacteriocin Genes
				3.3.1 DNA Extraction
				3.3.2 16S rRNA Gene PCR
				3.3.3 Bacteriocin Gene PCR
				3.3.4 DNA Sequencing and Analysis of the LAB Isolate´s 16S rRNA Gene
				3.3.5 DNA Sequencing and Analysis of the Bacteriocin Genes
		4 Notes
		References
Index