Molecular Typing in Bacterial Infections, Volume II

Contents
Contributors
Part I: Gastrointestinal Pathogens
	1: Campylobacter
		1.1	 Introduction
		1.2	 Early Typing Methods
		1.3	 Biochemical Tests
		1.4	 Phenotypic Methods
		1.5	 Enzyme-Based Methods
		1.6	 Pulsed-Field Gel Electrophoresis
		1.7	 Restriction Fragment Length Polymorphism of the flaA Gene (flaA-RFLP)
		1.8	 Commercial ELISA Kits
		1.9	 Molecular Methods
			1.9.1	 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry
			1.9.2	 PCR-Based Methods
			1.9.3	 Sequence-Based Methods
				1.9.3.1	 MLST
				1.9.3.2	 rMLST
				1.9.3.3	 cgMLST
				1.9.3.4	 wgMLST
			1.9.4	 Parallel Sequencing Methods
		1.10	 Concluding Remarks
		1.11	 Summary
		References
	2: Clostridioides difficile
		2.1	 Introduction
		2.2	 Virulence Factors
		2.3	 Epidemiology
		2.4	 Molecular Assays for C. difficile Detection
		2.5	 C. difficile Typing Methodologies
			2.5.1	 Restriction Endonuclease Analysis (REA)
			2.5.2	 Pulsed-Field Gel Electrophoresis (PFGE)
			2.5.3	 PCR Ribotyping
			2.5.4	 Multilocus Sequence Typing (MLST)
			2.5.5	 Repetitive Sequence-Based PCR Typing (Rep-PCR)
			2.5.6	 Toxinotyping
			2.5.7	 Multilocus Variable-Number Tandem-Repeat Analysis (MLVA)
			2.5.8	 Surface-Layer Protein A-Encoding Gene Typing (slpAST) as a Substitute for Serotyping
			2.5.9	 Whole-Genome Sequencing (WGS)
			2.5.10	 CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR))
			2.5.11	 DNA Microarray-Based Genotyping
		2.6	 Conclusions and Perspectives
		2.7	 Summary
		References
	3: Cronobacter
		3.1	 Introduction to the Cronobacter Genus
		3.2	 Cronobacter Pathogenicity and Virulence
			3.2.1	 Cronobacter Infections
			3.2.2	 Virulence Traits in Cronobacter spp.
		3.3	 Cronobacter in Food Industry
		3.4	 Cronobacter Isolation Methods in PIF and Other Foods
		3.5	 Cronobacter Identification Methods
		3.6	 Molecular Typing of Cronobacter Isolates
			3.6.1	 Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Cronobacter Strains
			3.6.2	 PCR-Based Serotyping of Cronobacter spp.
			3.6.3	 Capsular Profiling
			3.6.4	 Standardized Seven-Loci Multilocus Sequence Typing (MLST)
			3.6.5	 Ribosomal MLST (53 Loci)
			3.6.6	 Core-Genome MLST (1836 Loci)
			3.6.7	 CRISPR (Clustered Regularly Interspersed Short Palindromic Repeat)-Cas Array Profiling
			3.6.8	 Single-Nucleotide Polymorphism (SNP) Analysis
		3.7	 Summary
		References
	4: Oral and Intestinal Bacteroidetes
		4.1	 Introduction
		4.2	 The Genus Bacteroides
			4.2.1	 The Genus Bilophila
			4.2.2	 Bacteroides: Methods
				4.2.2.1	 Phenotypic Identification of Gram-Negative Anaerobic Saccharolytic Rods
				4.2.2.2	 Concept of PCR Fingerprinting
			4.2.3	 Bacteroides: Detailed Protocols
				4.2.3.1	 Bacteroides Strains, Culture Conditions, and DNA Extraction
				4.2.3.2	 PCR Amplification and Fingerprinting
			4.2.4	 Bacteroides: Results
				4.2.4.1	 PCR Fingerprinting
				4.2.4.2	 Characterization of Species and Establishment of Genetic Markers
				4.2.4.3	 Development of Specific PCR for Group Resp. Species Detection of B. fragilis
				4.2.4.4	 Amplification of the Enterotoxin Gene in B. fragilis Isolates
			4.2.5	 Bacteroides: Discussion
		4.3	 Porphyromonas: A Genus Becoming Diverse
			4.3.1	 Porphyromonas: Methods
				4.3.1.1	 The General Concept of ITS Determination
				4.3.1.2	 The Selection of Primers
				4.3.1.3	 The Sample Collection and DNA Extraction
			4.3.2	 Porphyromonas: Detailed Protocol
				4.3.2.1	 Bacterial Strains, Culture Conditions, and DNA Extraction
				4.3.2.2	 PCR Amplification and DNA Sequence Analysis
			4.3.3	 Porphyromonas: Results
			4.3.4	 Porphyromonas: Discussion
		4.4	 General Discussion and Final Remarks
		4.5	 Summary
		References
	5: Vibrio cholerae
		5.1	 Introduction
		5.2	 Background Information on V. cholerae
		5.3	 Polymerase Chain Reaction-Based Typing
			5.3.1	 Random Amplification of Polymorphic DNA (RAPD) Profiles
			5.3.2	 Other PCR-Based Typing Methods
			5.3.3	 Mobile Genetic Elements (MGEs)
			5.3.4	 Plasmids
			5.3.5	 Insertion Sequences (IS Elements)
			5.3.6	 Integrons and ICEs
		5.4	 Vibrio cholerae Pathogenicity Island and Vibrio Seventh Pandemic Islands
		5.5	 CTX Prophages
		5.6	 Multilocus Enzyme Electrophoresis
		5.7	 Ribotyping
		5.8	 Pulsed-Field Gel Electrophoresis
		5.9	 DNA Sequence-Based Typing Systems
			5.9.1	 Multilocus Sequence Typing
			5.9.2	 Multiple-Locus Variable Number of Tandem Repeat Analysis
		5.10	 Fingerprinting of Virulence Genes
		5.11	 Evidence Showing Intercontinental Spread of V. cholerae O1
		5.12	 Quorum-Sensing Systems
		5.13	 Microarray Analysis
		5.14	 Whole Genome Approach
		5.15	 Epilogue
		5.16	 Summary
		References
Part II: Pathogens Causing Healthcare-Associated Infections
	6: Acinetobacter baumannii
		6.1	 Introduction
		6.2	 Microbiology of Acinetobacter spp.
		6.3	 Taxonomy of Acinetobacter spp.
		6.4	 Clinical Significance of A. baumannii
		6.5	 Antibiotic Resistance
		6.6	 Epidemiology of Carbapenem-Resistant A. baumannii
		6.7	 Typing Methods for Acinetobacter spp.
			6.7.1	 Phenotypic Typing
			6.7.2	 Genotypic Typing
				6.7.2.1	 Band Pattern
				6.7.2.2	 Partial Sequencing
				6.7.2.3	 Whole Genome Sequencing
				6.7.2.4	 WGS Application
				6.7.2.5	 WGS Technology and Bioinformatics
		6.8	 Concluding Remarks and Recommendations
		6.9	 Summary
		References
	7: Enterococcus
		7.1	 Introduction
		7.2	 Characteristics and Current Classification of the Genus
		7.3	 Clinical Significance and Epidemiology
		7.4	 Resistance to Antimicrobial Agents
		7.5	 Typing Methods
			7.5.1	 Early Typing Methods
			7.5.2	 Molecular Typing Methods
		7.6	 Summary
		References
	8: Pseudomonas aeruginosa
		8.1	 Phenotyping Methods for P. aeruginosa
		8.2	 Restriction-Based Methods
			8.2.1	 Pulsed-Field Gel Electrophoresis (PFGE)
			8.2.2	 Restriction Fragment Length Polymorphism (RFLP)
			8.2.3	 Ribotyping
		8.3	 Amplified-Based Methods
			8.3.1	 Random Amplified of Polymorphic DNA (RAPD)
			8.3.2	 Amplified Fragment Length Polymorphisms (AFLP)
			8.3.3	 Multilocus Variable-Number Tandem Repeat (VNTR) Analysis (MLVA)
		8.4	 Sequencing-Based Method
			8.4.1	 Multilocus Sequence Typing (MLST)
			8.4.2	 Double-Locus Sequence Typing (DLST)
			8.4.3	 Whole Genome Sequencing (WGS)
		8.5	 Summary
		References
	9: Staphylococci
		9.1	 Introduction
		9.2	 Identification of Staphylococcal Species
		9.3	 MRSA Identification and SCCmec Typing
		9.4	 Historical Typing Methods
		9.5	 Current Molecular Typing Methods
			9.5.1	 Pulsed-Field Gel Electrophoresis (PFGE) Typing
			9.5.2	 Staphylococcal Protein A (spa) Typing
			9.5.3	 Multilocus Sequence Typing (MLST)
			9.5.4	 Microarray
		9.6	 Whole Genome Sequencing (WGS) and the Future of MRSA Typing
		9.7	 Conclusions
		9.8	 Summary
		References
Part III: Vector-Borne and Biosafety Level 3 Pathogens
	10: Bartonellaceae
		10.1	 Introduction
		10.2	 Phenotypic Techniques
		10.3	 Molecular Techniques
			10.3.1	 Species Identification
				10.3.1.1	 16S-Based Techniques
				10.3.1.2	 Amplified Ribosomal DNA Restriction Analysis
				10.3.1.3	 Ribotyping
				10.3.1.4	 DNA-DNA Hybridization
				10.3.1.5	 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
				10.3.1.6	 Specific PCRs
			10.3.2	 Clonal Determination
				10.3.2.1	 Multilocus Sequence Typing
				10.3.2.2	 Multispacer Typing
				10.3.2.3	 Multilocus Variable Copy Numbers of Tandem Repeats Analysis
				10.3.2.4	 Pulsed-Field Gel Electrophoresis
		10.4	 Whole Genomic Sequencing
		10.5	 PCR Typing-Based Techniques
			10.5.1	 Amplified Fragment Length Polymorphism
			10.5.2	 Repetitive Extragenic Palindromic PCR
			10.5.3	 Enterobacterial Repetitive Intergenic Consensus PCR
			10.5.4	 Randomly Amplified Polymorphic DNA PCR
			10.5.5	 16S rRNA Type-Specific Amplification
			10.5.6	 Infrequent Restriction Site PCR
		10.6	 Summary
		References
	11: Brucella
		11.1	 Introduction
			11.1.1	 Brucellosis
			11.1.2	 The Genus Brucella
			11.1.3	 Significance of Molecular Typing Approaches to Brucella Control and Eradication
		11.2	 PCR-Based Molecular Detection Methods
			11.2.1	 Brucella Genus-Specific PCR Targets: bcsp31
			11.2.2	 Brucella Genus-Specific PCR Targets: IS711
			11.2.3	 Brucella Genus-Specific PCR Targets: 16S rRNA, omp, per and Others
			11.2.4	 Application of PCR to Clinical Samples
		11.3	 Early Approaches to Molecular Typing
		11.4	 Molecular Typing Assays Discriminating Between Brucella Species
			11.4.1	 Single Loci Discriminating Between Species
			11.4.2	 Multiplex Conventional PCR Approaches Discriminating Between Species
			11.4.3	 Targeted Real-Time Assays
			11.4.4	 Single-Species Assays
			11.4.5	 Real-Time PCR Assays Based on Canonical SNPs
			11.4.6	 High-Resolution Melt (HRM) Analysis Assays Based on Canonical SNPs
			11.4.7	 Vaccine Strain-Specific Assays
		11.5	 Approaches to Sub-Species Molecular Typing
			11.5.1	 Multi-Locus Sequence Typing (MLST)
			11.5.2	 Multi-Locus Variable Number Tandem Repeat Analysis (MLVA)
		11.6	 Whole-Genome Sequencing-Based Approaches
			11.6.1	 Early Whole-Genome Sequencing Studies
			11.6.2	 High-Resolution Strain Typing Using Whole-Genome Sequencing
			11.6.3	 Genome-Scale MLST Approaches (cgMLST and wgMLST)
			11.6.4	 Whole-Genome SNP Approaches
			11.6.5	 In Silico Extraction of Data for Existing Typing Schemes
		11.7	 Conclusions and Future Directions
		References
	12: Coxiella burnetii
		12.1	 Introduction
		12.2	 The Pathogen
		12.3	 Molecular Typing Methods
			12.3.1	 History – Typing Is an “Old” Science in Q Fever
			12.3.2	 Today – Reduced to the Max – A Lot of Epidemiological Studies – No Use in Routine Diagnostics
			12.3.3	 On the Edge to Tomorrow – Whole Genome Sequencing and Enrichment Techniques
			12.3.4	 Database Issues
		12.4	 Conclusions
		References
Index