Springer principles of bacterial detection biosensors recognition receptors and microsystems sep 2008 ISBN 0387751122 pdf

MicroSERS for the Detection and Identification of Pathogens and Toxins .... Rapid Nucleic Acid-Based Diagnostics Methods for the Detectionof Bacterial Pathogens Barry Glynn 1.. Rapid Nuc

Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems

Principles of Bacterial Detection: Biosensors, Recognition

Receptors and Microsystems

Mohammed Zourob Souna Elwary

Biophage Pharma Inc Consultant to Biophage Pharma Inc

Library of Congress Control Number: 2007941938

© 2008 Springer Science+Business Media, LLC

All rights reserved This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.

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Bacterial contamination of food and water resources, as well as the increasing incidence ofnosocomial infections, has us on our toes, looking for ways of recognizing these elements Inaddition, the recent and growing threats to personal and territorial securities make this task evenmore urgent Therefore, accurate assessment of the state of current technologies is a prerequisitefor undertaking any course of action towards future improvements In particular, development

of new detection and identification technologies for the plethora of bacterial agents has becomeincreasingly important to scientists and to regulatory agencies In recent years, there has beenmuch progress in the field of bacterial agents detection, resulting in the development of moreaccurate, fast, analyte-specific, robust, and cost effective techniques by incorporating emergingtechnologies from various disciplines

Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems

presents a significant and up-to-date review of various integrated approaches for bacterialdetection by distinguished engineers and scientists This work is a comprehensive approach tobacterial detection, presenting a thorough knowledge of the subject and an effective integration

of disciplines in order to appropriately convey the state-of the-art fundamentals and applications

of the most innovative approaches

The book consists of four parts The first part (Chapters 1–4) is an introduction topathogenic bacteria and sampling techniques and provides an overview of the rapid microbio-logical methods The second part (Chapters 5–20) describes the different transducers used forbacterial detection It covers the theory behind each technique and delivers a detailed state-of-the-art review for all the new technologies used The third part (Chapters 21–29) coversthe different recognition receptors used in the latest methods for the detection of bacteria

It describes in detail the use of immunoassays, nucleic acids, oligonucleotide microarrays,carbohydrates, aptamers, protein microarrays, bacteriophage, phage display, and molecularimprinted polymers as recognition elements The fourth part (Chapters 30–36) covers thedifferent microsystems used for detection/identification and bacterial manipulations, mainlybacteria lysis in microfluidics, PCR in microfluidics, dielectrophoresis, ultrasonic manipulationtechniques, and mass spectrometry

We anticipate that the book will be helpful to academicians, practitioners, andprofessionals working in various fields, including biomedical sciences, physical sciences,microsystems engineering, nanotechnology, veterinary science and medicine, food QA, bioter-rorism and security as well as allied health, healthcare and surveillance Since the fundamentalsare also reviewed, we believe that the book will appeal to advanced undergraduate and graduatestudents who study in areas related to bacterial detection

We gratefully acknowledge all authors for their participation and contributions, whichmade this book a reality We give many thanks to Olivier Laczka and Joseph Piliero for thebook cover design

Mohammed ZourobSouna ElwaryAnthony TurnerJune 2008

v

Part I Introduction

1 Introduction to Pathogenic Bacteria

Tracey Elizabeth Love and Barbara Jones

1 Pathogenic Microorganisms 3

1.1 Toxins 4

1.2 Adherence 4

1.3 Invasion 7

1.4 Evasion of the Host Immune Response 7

1.5 Iron Acquisition 8

1.6 Regulation of Virulence Factors 8

2 Sources and Routes of Infection 9

2.1 Natural Infection 9

2.2 Food and Water 9

2.3 Hospital Acquired Infections 10

2.4 Intentional Infection—Biological Warfare 10

3 Detection of Pathogenic Microorganisms 11

4 Conclusions 12

References 12

2 Sample Preparation: An Essential Prerequisite for High-Quality Bacteria Detection Jan W Kretzer, Manfred Biebl and Stefan Miller 1 Introduction 15

2 The Sample 16

3 Sampling 17

3.1 Sample drawing 17

4 Microbiological Examination of Foods 17

5 Microbiological Examination of Surfaces 17

6 Microbiological Examination of Air 18

7 Sample Handling 20

8 Sample Preparation 21

9 Sample Preparation for Detection of Intact Bacterial Cells 21

10 Sample Preparation for Detection of Bacterial Nucleic Acids 23

11 Conclusions and Future Perspectives 27

References 28

3 Detection of Bacterial Pathogens in Different Matrices: Current Practices and Challenges Ahmed E Yousef 1 Introduction 31

2 Analytical Tools and Methods: A Historical Perspective 32

vii

3 Defining the Terms 32

4 Matrix Complexity and Pathogen Detection 32

5 Techniques Currently Used in Pathogen Detection Methods 33

5.1 Culture Techniques 33

5.2 Enzyme-Linked Immunoassay 35

5.3 Polymerase Chain Reaction (PCR) 36

6 Basics of Pathogen Detection 36

6.1 Sampling 37

6.1.1 Air Sampling 37

6.1.2 Surfaces Sampling 37

6.1.3 Bulk Sampling 39

6.2 Sample Preparation 39

6.3 Pathogen Amplification 39

6.4 Selection and Screening 40

6.5 Identification 40

6.5.1 Morphological Characteristics 41

6.5.2 Biochemical and Physiological Traits 41

6.5.3 Serological Properties 42

6.5.4 Genetic Characteristics 42

6.6 Pathogenicity Testing 43

6.6.1 Koch’s Postulates 43

6.6.2 Mammalian Cell Culture (Tissue Culture) 43

6.6.3 Virulence Genes and Gene Expression Products 44

6.7 Testing for Specific Traits 44

7 Challenges to Current Detection Methods 44

7.1 Pathogen Quantification Problems 44

7.2 Can a Small Bacterial Population be Detected Rapidly and Reliably? 44

7.3 Which Traits to Analyze, and How Many Tests are Needed for Identifying a Bacterial Pathogen? 45

7.4 Real-Time Detection 46

References 46

4 Overview of Rapid Microbiological Methods Jeanne Moldenhauer 1 Introduction 49

2 A History of Rapid Microbiological Methods: Industry Reluctance to Accept These Methods 50

3 Types of Microbial Testing Performed 50

4 Types of Rapid Microbiological Methods 50

4.1 Growth-Based Technologies 50

4.2 Viability-Based Technologies 50

4.3 Cellular Component or Artifact-Based Technologies 51

4.4 Nucleic Acid-Based Technologies 51

4.5 Automated Methods 51

4.6 Combination Methods 51

5 Overview of Rapid Technologies and How They Work 51

5.1 Adenosine Tri-Phosphate (ATP) Bioluminescence 51

5.2 Adenylate Kinase 52

5.3 Autofluorescence 52

5.4 Biochemical Assays and Physiological Reactions 52

5.5 Biosensors and Immunosensors 53

5.6 Carbon Dioxide Detection 53

5.7 Changes in Headspace Pressure 53

5.8 Colorimetric Detection of Carbon Dioxide Production 53

5.9 Concentric Arcs of Photovoltaic Detectors with Laser Scanning 54

5.10 Direct Epifluorescent Filter Technique (DEFT) 54

5.11 DNA Sequencing 54

5.12 Endospore Detection 55

5.13 Enzyme Linked Immunosorbent Assay (ELISA) 55

5.14 Flow Cytometry 55

5.15 Fluorescent Probe Detection 55

5.16 Fatty Acid Profiles (Fatty Acid Methyl Esters, FAMEs) 56

5.17 Fourier Transformed Infrared Spectroscopy (FTIR) 56

5.18 Gram Stains (Rapid Method) 56

5.19 Impedance 57

5.20 Immunological Methods 57

5.21 Lab-on-a-Chip (LOC), Arrays, Microarrays and Microchips 57

5.22 Limulus Amebocyte Lysate (LAL) Endotoxin Testing 58

5.23 Mass Spectrometry (Matrix-Assisted Laser Desorption-Time of Flight (MALTI-TOF)) 58

5.24 Microcalorimetry 58

5.25 Micro-Electro-Mechanical Systems (MEMS) 59

5.26 Nanotechnology 59

5.27 Near Infrared Spectroscopy (NIRS) 59

5.28 Nucleic Acid Probes 59

5.29 Optical Particle Detection 59

5.30 Polymerase Chain Reaction (PCR) 60

5.31 Rep-PCR 60

5.32 Raman Spectroscopy 61

5.33 Ribotyping/Molecular Typing 61

5.34 Solid Phase Laser Scanning Cytometry 61

5.35 Southern Blotting/Restriction Fragment Length Polymorphism 62

5.36 Spiral Plating 62

5.37 Turbidimetry 62

6 Potential Areas of Application of Rapid Microbiological Methods 62

7 Disclaimer 75

8 Conclusions 75

References 75

Part II Biosensors 5 Surface Plasmon Resonance (SPR) Sensors for the Detection of Bacterial Pathogens Allen D Taylor, Jon Ladd, Jiˇrí Homola and Shaoyi Jiang 1 Introduction 83

2 Fundamentals of Surface Plasmon Resonance Biosensing 83

3 SPR Sensor Instrumentation 85

4 Surface Chemistries and Molecular Recognition Elements 88

5 Detection Formats 90

6 Quantification of Bacteria Cells 91

6.1 Challenges for the Detection of Whole Bacteria by SPR 91

6.2 Effect of Bacteria Sample Treatment 92

6.3 Examples of Bacteria Detection 92

6.3.1 Escherichia coli 93

6.3.2 Salmonella spp . 97

6.3.3 Listeria monocytogenes 98

6.3.4 Other Bacteria 98

6.3.5 Detection of Multiple Bacteria 99

7 Genetic Markers 101

8 Antibody Biomarkers 103

9 Conclusions and Future Perspectives 103

References 104

6 Bacterial Detection Using Evanescent Wave-Based Fluorescent Biosensors Kim E Sapsford and Lisa C Shriver-Lake 1 Introduction 109

2 Current State of Bacterial Fluorescent TIRF Biosensors 112

2.1 Non-Planar Substrates 112

2.1.1 Fiber Optics 112

2.1.2 Capillaries 112

2.2 Planar Substrates 112

2.2.1 NRL Array Biosensor 113

2.2.2 Other Optical Waveguides 115

2.2.3 TIRF-Microscopy 116

3 Future Aspects of Bacterial Fluorescent TIRF Biosensors 117

4 Conclusions 119

References 120

7 Fiber Optic Biosensors for Bacterial Detection Ryan B Hayman 1 Fiber Optic Biosensors 125

1.1 Whole-Cell Detection 126

1.1.1 Evanescent-Field Sensing 126

1.1.2 Sandwich Immunoassays 127

1.2 Bead-Based Arrays 128

1.3 Nucleic Acid Sandwich Assays 129

1.4 Nucleic Acid Direct Hybridization 131

1.5 Extension Reactions 134

2 Conclusions and Future Perspectives 134

References 135

8 Integrated Deep-Probe Optical Waveguides for Label Free Bacterial Detection Mohammed Zourob, Nina Skivesen, Robert Horvath, Stephan Mohr, Martin B McDonnell and Nicholas J Goddard 1 Introduction 139

1.1 Planar Optical Waveguides 141

1.2 Total Internal Reflection and Evanescent Waves 141

1.3 Waveguide Modes 143

1.4 Frustrated Total Internal Reflection, Leaky Modes 144

1.5 Literature on Waveguides for Bacterial Detection 144

2 Deep-Probe Optical Waveguide Sensors with Tunable Evanescent Field 145

2.1 Waveguide Modes, Light Coupling and Sensing Depths of Evanescent Waves 146

2.1.1 Light Coupling Techniques 148

2.2 Waveguide Designs Based on Low-Index Substrates 150

2.2.1 Bacteria Detection Using Reverse Symmetry Waveguides 151

2.3 Waveguide Designs Based on Metal- and Dye-Clad Substrates—Leaky Modes 152

2.3.1 Results 156

3 Integrated Deep-Probe Optical Waveguides Systems 160

3.1 Integration with Electric Field 161

3.2 Integration with Ultrasound Standing Waves (USW) 163

4 Conclusions and Future Perspectives 166

References 166

9 Interferometric Biosensors Daniel P Campbell 1 Principles of Optical Interferometry 169

1.1 Optical Waveguides 171

1.2 Planar Waveguide Operation 172

1.3 Types of Waveguides 175

2 Light Coupling Methods 178

2.1 Interferometers 180

2.2 Collinear or Single Channel Interferometers 183

2.3 Two-Channel Interferometers 186

3 Interferometric Array Sensors 192

4 Surface Plasmon Interferometry 195

5 Other Interferometric Methods and Designs 196

6 Surface Functionalization 197

7 Sample Collection Systems 198

8 Interferometric Applications for Whole-Cell Detection 199

9 Advantages and Limitations 206

10 Potential for Improving Current Performance 206

References 208

10 Luminescence Techniques for the Detection of Bacterial Pathogens Leigh Farris, Mussie Y Habteselassie, Lynda Perry, S Yanyun Chen, Ronald Turco, Brad Reuhs and Bruce Applegate 1 Beyond Robert Boyle’s Chicken 214

2 The Bacterial (lux) Luminescent System for Direct Pathogen Detection 215

3 The Firefly (luc) Luminescent System for Direct Pathogen Detection 219

4 The Use of Alternative Luciferases in Pathogen Detection 222

5 Luminescent-Based Immunoassays 222

6 Chemiluminescence Detection Methods 222

7 Conclusions and Future Perspectives 225

References 226

11 Porous and Planar Silicon Sensors Charles R Mace and Benjamin L Miller 1 Introduction 231

1.1 Porous Silicon: A Three-Dimensional Matrix for Biosensing 232

1.2 Effect of PSi Immobilization on Probe Viability: Experiments with GST 233

1.3 Toward Larger Targets: The First Macroporous Microcavity Structures 235

1.4 Porous Silicon Bandgap Sensors in Novel Formats: “Smart Bandages” and “Smart Dust” 235

2 Arrayed Imaging Reflectometry—A Planar Silicon Biosensor 236

2.1 Theory 236

2.1.1 Physical Rationale 236

2.1.2 Substrate Design 237

2.1.3 Mathematical Model 238

2.1.4 Monitoring the Null Reflectance Condition 240

2.2 Applications of AIR Biosensing 242

2.2.1 Limitations 242

2.2.2 Probe Immobilization 244

2.2.3 Pathogen Detection 246

3 Conclusions and Future Perspectives 250

References 251

12 Acoustic Wave (TSM) Biosensors: Weighing Bacteria Eric Olsen, Arnold Vainrub and Vitaly Vodyanoy 1 Introduction 255

2 Historical Perspective, Theory and Background 256

2.1 Piezoelectricity and Acoustic Waves 256

2.2 Acoustic Wave Devices 256

3 TSM Biosensors 259

3.1 Detection of Microorganisms 261

3.2 Measurement in Liquid 263

3.3 TSM Biosensor Characteristics 264

3.4 Commercial TSM Microbalances 267

3.5 Immobilization of Probes onto Sensor Surface 269

3.5.1 Physical Adsorption 271

3.5.2 Other Coupling Methods 272

3.5.3 Combined Langmuir-Blodgett/Molecular Assembling Method 272

3.5.4 Solvent-Free Purified Monolayers 275

3.5.5 Immobilization of Monolayers of Phage Coat Proteins 276

3.5.6 Immobilization of Molecular Probes onto Porous Substrates 281

4 Problem of “Negative Mass” 282

5 Coupled Oscillators Model 286

6 Conclusions 290

References 291

13 Amperometric Biosensors for Pathogenic Bacteria Detection Ilaria Palchetti and Marco Mascini 1 Introduction 299

2 Amperometric Biosensors 300

2.1 Microbial Metabolism-Based Biosensors 302

2.2 Immunosensors 303

2.3 DNA-Based Biosensors 306

3 Conclusion and Future Perspectives 310

References 310

14 Microbial Genetic Analysis Based on Field Effect Transistors Yuji Miyahara, Toshiya Sakata and Akira Matsumoto 1 Introduction 313

2 Fundamental Principles of Field Effect Devices 314

2.1 Metal-Insulator-Semiconductor (MIS) Capacitor 314

2.2 Principles of Biologically Coupled Field Effect Transistors for Genetic Analysis (Genetic FETS) 315

3 Fundamentals of Genetic Analysis 317

3.1 DNA 317

3.2 Genetic Analysis 317

3.3 DNA Chip / DNA Microarray 318

4 Immobilization of DNA Molecules on the Surfaces of Solid Substrates 318

4.1 Silanization 318

4.2 Thiol-Gold Bonding 320

4.3 Avidin, Streptavidin and Biotin 320

4.4 Others 321

5 Genetic Analysis Based on Field Effect Devices 322

5.1 Fundamental Characteristics of Genetic Field Effect Devices 322

5.1.1 Detection of DNA Molecular Recognition Events 322

5.1.2 Immobilization Density of Oligonucleotide Probes 326

5.2 Single Nucleotide Polymorphisms (SNPs) Analysis 327

5.2.1 Controlling Hybridization Temperature for SNPs Analysis 328

5.2.2 SNPs Analysis Based on Primer Extension 329

5.3 DNA Sequencing 331

6 Conclusions and Future Perspectives 335

References 336

15 Impedance-Based Biosensors for Pathogen Detection Xavier Muñoz-Berbel, Neus Godino, Olivier Laczka, Eva Baldrich, Francesc Xavier Muñoz and Fco Javier Del Campo 1 Introduction 341

2 Fundamentals of Electrochemical Impedance Spectroscopy 342

2.1 Data Analysis: Plotting 344

2.2 Data Analysis: Interpretation 344

2.2.1 Non-Faradaic Parameters 345

2.2.2 Faradaic Parameters 347

2.3 Measuring at Impedimetric Biosensors 350

2.3.1 Measurement Modes 350

2.4 Bacterial Parasitizing Effect on Electrode Surface 353

3 Development of an Immunosensor 354

3.1 Biological Recognition Elements in Biosensors for Pathogen Detection 354

3.1.1 Antibodies 355

3.1.2 Nucleic Acids 355

3.1.3 Aptamers 356

3.1.4 Other Recognition Strategies 356

3.2 Surface Modification Methods 357

3.2.1 Adsorption 357

3.2.2 Self-assembled Monolayers 358

3.2.3 Silanisation 359

3.2.4 Protein A and Protein G 360

3.2.5 The Biotin-(Strept)Avidin System 360

3.2.6 Chemical Conjugation 361

3.2.7 Entrapment 362

3.2.8 Microencapsulation 362

3.3 Blocking 362

3.4 Signal Amplification 363

3.5 The Need for Negative Controls 364

3.6 Development of Novel Strategies: Assessing Performance Using ELISA and Microscopy 365

4 Current EIS Biosensors for Pathogen Detection 365

4.1 Biosensors Based on Interfacial Capacitance Changes 366

4.2 Biosensors Based on Charge-Transfer Resistance Changes 367

4.3 Biosensors Based on Conductivity Changes 369

4.4 Other Approaches 370

5 Conclusions and Future Perspectives 370

References 371

16 Label-Free Microbial Biosensors Using Molecular Nanowire Transducers Evangelyn Alocilja and Zarini Muhammad-Tahir 1 Introduction 377

1.1 Rationale for Rapid Tests 377

1.2 Target Microorganisms and Matrices 378

1.2.1 Escherichia coli 378

1.2.2 Salmonella 379

1.2.3 Bovine Viral Diarrhea Virus 380

1.3 Food Safety Applications 381

2 Biosensor Formats 382

2.1 Definition 382

2.2 Antibodies as Biological Sensing Element 382

2.3 DNA as Biological Sensing Element 384

2.4 DNA-Based Biosensors 385

2.5 Antibody-Based Biosensors 387

2.6 Biosensor Transducing Element: Conducting Polymer 388

2.6.1 Polyaniline 390

2.6.2 Self-doped Polyaniline 391

2.6.3 Carbon Nanotubes 391

2.7 Conducting Polymer-Based Biosensor for Microbial/Viral Detection 392

3 Illustration: Biosensor Using Self-doped and Non-self-doped Pani 392

3.1 Pani Preparation 392

3.2 Pani Characterization 392

3.2.1 Conductivity Measurement 392

3.2.2 Biosensor Fabrication 393

3.2.3 Indium Tin Oxide/Pani Biosensor 393

3.2.4 Lateral Flow Conductometric Biosensor 393

3.2.5 Signal Measurement 393

3.3 Properties of Pani 394

3.4 Detection Concept of the Biosensor 398

3.5 Biosensor Properties 399

3.5.1 ITO-Pani Biosensor 399

3.6 Lateral Flow Conductometric Biosensor 403

3.7 Biosensor Performance 404

3.7.1 ITO/Pani Biosensor 404

3.8 Conductometric Biosensor 404

4 Conclusions and Future Perspectives 406

References 406

17 Magnetic Techniques for Rapid Detection of Pathogens Yousef Haik, Reyad Sawafta, Irina Ciubotaru, Ahmad Qablan, Ee Lim Tan and Keat Ghee Ong 1 Introduction 415

2 Synthesis of Magnetic Particles 417

2.1 Effect of Particle Size 418

2.2 Synthesis Techniques 423

2.3 Encapsulation of Magnetic Particles 423

2.3.1 Methods of Preparing Polymer/Protein Coatings 424

2.3.2 Examples of Polymer/Protein Encapsulated Particles 426

3 Immobilization Strategies 426

3.1 Modification of Particle Surface with a Ligand 430

4 Biological Targets 430

5 Magnetic Immunoassays 430

5.1 Direct Immunoassay Detection Using Magnetic Beads 430

5.1.1 Superconducting Quantum Interference Devices 431

5.1.2 ABICAP Column 432

5.2 Indirect Immunoassay Detection Using Magnetic Beads 433

5.2.1 ELISA 433

6 Handling Techniques 438

7 Magnetic Separation 439

7.1 Magnetic Force 439

7.2 High-Field Electromagnets 440

7.3 Permanent Magnets 441

7.4 Numerical Analysis for Permanent Magnet Arrangements 442

8 Giant Magnetoresistive (GMR) Devices for Bacterial Detection 446

9 Bacteria Detection with Magnetic Relaxation Signal 448

10 Magnetoelastic Sensors for Bacterial Detection 449

10.1 E coli Detection 450

11 Conclusions and Future Perspectives 453

References 454

18 Cantilever Sensors for Pathogen Detection Raj Mutharasan 1 Introduction 459

2 Millimeter-Sized Cantilever Sensors 460

3 Reported Work on Detecting Cells Using Cantilever Sensors 461

4 Physics of Cantilever Sensors 463

5 Resonance Modes 466

6 Characterization of PEMC Sensors 468

7 Mass Change Sensitivity 468

8 Antibody Immobilization Methods 469

9 Detection in Batch and Stagnant Samples 470

10 Detection in Flowing Samples 473

11 Selectivity of Detection 475

12 Conclusions 477

References 478

19 Detection and Viability Assessment of Endospore-Forming Pathogens Adrian Ponce, Stephanie A Connon and Pun To Yung 1 Introduction 481

1.1 Historical Perspective 481

1.2 Endospore Dormancy, Resistance and Longevity 482

1.3 Endospores as Biodosimeters for Evaluating Sterilization Regimes 484

1.4 Endospore-Forming Pathogens 485

1.5 Bioweapons, Bioinsecticides and Probiotics 487

2 Detection of Endospore-Forming Pathogens and their Endospores 489

2.1 Phenotypic Identification 489

2.1.1 Phenotypic Identification of Bacillus anthracis 490

2.1.2 Phenotypic Identification of Clostridium perfringens 490

2.2 Parameters of a Sensor 491

2.3 Rapid Immunoassays 492

2.3.1 Enzyme-Linked Immunosorbent Assays 492

2.3.2 Lateral-Flow Immunoassays 493

2.3.3 Immunomagnetic Electrochemiluminescence 493

2.3.4 Flow Cytometry 494

2.3.5 Vegetative Cells 494

2.4 Rapid Nucleic Acid Assays 495

2.4.1 PCR Sample Preparation and Endospore Lysis 495

2.4.2 The PCR Reaction 496

2.4.3 Specificity of PCR Primers for Bacillus anthracis Detection 496

2.4.4 Rapid PCR Detection Methods High Throughput and real-time PCR 497

2.4.5 Field Implementation of Rapid PCR for Analysis of Environmental Samples 498

2.4.6 Monitoring the Air for Bacillus anthracis Endospores by PCR 500

2.5 Rapid Detection of Endospores via Dipicolinic Acid Biomarker 501

2.5.1 Terbium Dipicolinic Acid Luminescence Assay 501

2.5.2 Anthrax Smoke Detector 503

3 Validation of Sterilization by Rapid Endospore Viability Assessment 505

3.1 Measuring Endospore Viability and Inactivation 505

3.2 Measuring Endospore Inactivation using Germinability Assays 508

3.2.1 Rapid Germinability Assays 508

3.2.2 Nucleic Acid-Based Amplification Methods for Detecting Germinable, Viable Bacillus anthracis Spores 508

3.2.3 Germination Observed via Loss of Phase Brightness 509

3.2.4 Germination Observed via DPA release 510

3.3 Measuring Endospore Inactivation Using Metabolic Activity Assays 512

4 Conclusions and Future Perspectives 513

References 514

20 Label-Free Fingerprinting of Pathogens by Raman Spectroscopy Techniques Ann E Grow 1 Introduction 525

2 Raman Microscopy for Whole-Organism Fingerprinting 527

3 Surface-Enhanced Raman Scattering (SERS) for Whole-Organism Fingerprinting 531

4 MicroSERS for the Detection and Identification of Pathogens and Toxins 534

4.1 MicroSERS Detection of Bacteria 535

4.1.1 SERS Fingerprinting of Bacteria 535

4.1.2 Impact of Growth Conditions on Bacterial Fingerprints 536

4.1.3 Viable vs Nonviable Bacteria 539

4.1.4 Integrated MicroSERS Detection and Identification of Bacteria 542

4.1.5 Impact of Growth Conditions on Biomolecule Capture 544

4.1.6 Analysis of Bacteria in Complex Samples 544

4.2 MicroSERS Detection of Spores 545

4.2.1 SERS Fingerprinting of Spores 545

4.2.2 Impact of Growth Conditions on Spore Fingerprints 547

4.2.3 Viable vs Nonviable Spores 550

4.2.4 Integrated MicroSERS Detection and Identification of Spores 551

4.3 MicroSERS Detection of Bacterial Toxins 554

4.3.1 SERS Fingerprinting of Toxins 555

4.3.2 Analysis of Toxins in Complex Samples 558

5 Conclusion and Future Perspectives 559

References 560

Part III Recognition Receptors 21 Antibodies and Immunoassays for Detection of Bacterial Pathogens Padmapriya P Banada and Arun K Bhunia 1 Introduction 567

2 Antibodies 568

2.1 Polyclonal Antibody 570

2.2 Monoclonal Antibody 570

2.3 Use of Synthetic Peptides for Antibody Production 571

2.4 Recombinant DNA Technology 573

2.4.1 Phage Display 573

3 Capture and Concentration of Cells by Immunomagnetic Separation 575

3.1 Automated IMS Systems 577

4 Immunoassays for Pathogen Detection 577

4.1 Radioimmunoassay 577

4.2 Enzyme Immunoassays 577

4.2.1 Escherichia coli 580

4.2.2 Listeria monocytogenes 583

4.2.3 Salmonella 583

4.2.4 Staphylococcal Enterotoxins 583

4.2.5 Clostridium botulinum Toxins 584

4.3 Lateral Flow Immunoassay 584

4.4 Other Immunoassays 585

4.4.1 Latex Agglutination (LA) and Reverse Passive Latex Agglutination (RPLA) Tests 585

4.4.2 Enzyme-Linked Fluorescent Assay 585

4.4.3 Time-Resolved Fluorescence Immunoassay 585

4.4.4 Chemiluminescent Immunoassay 586

4.4.5 Capillary Microbead (Spheres) Immunoassay 586

4.4.6 Electrochemical-Immunoassay 586

4.5 Optical Biosensors 587

4.5.1 Surface Plasmon Resonance 587

4.5.2 Fiber-Optic Biosensors 587

4.5.3 Antibody-Based Microfluidic Sensors 588

4.5.4 Serodiagnosis 589

5 Recent Developments in Immunoassays 590

5.1 Protein/Antibody Microarrays 590

5.2 Mass Spectrometric Immunodetection 591

5.3 SERS Biochip Technology 591

6 Limitations and Challenges 591

6.1 Specificity and Sensitivity 591

6.2 Effect of Physical and Chemical Stresses on the Expression Profile of Antigens in Bacteria 592 6.2.1 Effect of Media Composition on the Expression of Proteins in Bacteria 592

6.2.2 Effect of Stress on the Expression of Proteins in Bacteria 593

7 Conclusions and Future Perspectives 594

References 595

22 Rapid Nucleic Acid-Based Diagnostics Methods for the Detection

of Bacterial Pathogens

Barry Glynn

1 Introduction 603

1.1 Detection of Pathogenic Bacteria from Clinical Samples 604

1.2 NAD Assays for the Detection of Respiratory Infection, Sepsis and Sexually Transmitted Infection 604

1.3 Profiling of Multi-drug Resistance 606

1.4 Bioterrorism 606

2 Detection of Bacterial Food-Borne Pathogens 606

2.1 Recent Outbreaks 606

2.2 Benefits and Limitations of Conventional Methods 607

2.3 Development of Rapid Diagnostics Methods 607

3 Rapid Nucleic Acid Diagnostics for Bacterial Food-Borne Pathogens 607

3.1 In Vitro Nucleic Acid Amplification-Based Detection of Food-Borne Pathogens 607

3.2 Requirements for a NAD-Based Food Assay 608

3.3 Polymerase Chain Reaction (PCR) 608

3.4 Application of PCR-Based Tests to Pathogen Detection in Food Samples 609

3.5 Use of RNA as an Alternative Nucleic Acid Diagnostic Target 610

3.6 Sample Preparation for NAD from Clinical Sample Types 611

3.7 Limitations of NAD in Clinical Settings 611

4 Formats of NAD Assays for Food Pathogen Detection 612

4.1 Nucleic Acid-Based Diagnostics Based on In Vitro Amplification Technologies 612

4.2 PCR-ELISA and PCR-DNA Probe Membrane Based Assays for Campylobacter and Salmonella 612

4.3 Specific Examples of Nucleic Acid Diagnostics Assays for the Detection of Bacterial Food-Borne Pathogens 613

4.3.1 Commercially Available Conventional NAD Assays for Food-Borne Bacterial Pathogens 614

4.3.2 Alternative In Vitro Amplification Technologies 615

4.4 Standardisation of In Vitro Amplification-Based NAD Assays and Inter-Laboratory Validation Studies 616

4.5 Real-Time In Vitro Amplification-Based Nucleic Acid Diagnostics 617

4.5.1 Specific Examples of Real-Time PCR Assays for the Detection of Bacterial Food-Borne Pathogens 617

4.5.2 Alternative Real-Time In Vitro Amplification-Based Diagnostics Technologies 618

4.6 Limitations and Other Considerations for In Vitro Amplification NAD Tests 619

4.7 Non-Amplified Direct DNA Probe-Based Nucleic Acid Diagnostics 620

4.8 DNA-Probe Based Detection Methods 620

5 Conclusions and Future Perspectives 621

5.1 Emerging Nucleic Acid Diagnostic Technologies for Food-Borne Pathogen Detection 621

5.1.1 Biosensors 621

5.1.2 Microarrays 622

References 623

23 Oligonucleotide and DNA Microarrays: Versatile Tools for Rapid Bacterial Diagnostics Tanja Kostic, Patrice Francois, Levente Bodrossy and Jacques Schrenzel 1 Introduction 629

2 Microarray Technology 630

3 Technical Aspects of Microarray Technology 632

3.1 Probes 632

3.1.1 Genome Fragments 632

3.1.2 PCR Products 632

3.1.3 Oligonucleotide Probes 632

3.2 Substrates for Printing 634

3.2.1 Slides with Poly-L-Lysine Coating 634

3.2.2 Slides with Amino Silane Coating 635

3.2.3 Slides with Aldehyde Coating 635

3.2.4 Slides with Epoxy Coating 635

3.2.5 Proprietary Surface Chemistries 636

3.2.6 Probe Spacers 636

3.3 Targets for Microarray Analysis 637

3.3.1 Target Amplifications and Sensitivity Issues 637

3.3.2 Labeling of the Targets 638

3.3.3 Hybridization and Wash Conditions 638

3.4 Classical Commercially-Available Microarray Formats 639

3.4.1 Spotting Approaches 639

3.4.2 In Situ Synthesis 639

3.5 Alternative Methods for Improving Microarray-Based Detection Sensitivity 641

3.5.1 Resonance-Light Scattering (RLS) 641

3.5.2 Planar-Waveguide Technology (PWT) 641

3.5.3 Liquid Arrays 641

3.5.4 Three-Dimensional Microarray Formats 642

3.6 Marker Genes Used on Microbial Diagnostic Microarrays (MDMs) 643

4 Analysis and QC Aspects 643

5 Applications of Microarray Technology in Microbial Diagnostics 644

5.1 Gene Expression Studies 644

5.2 Comparative Genome Hybridizations (CGH) 645

5.3 Generic or Universal Microarrays 646

5.4 Microarrays for Sequence Analysis 647

5.5 Microbial Diagnostic Microarrays 648

6 Conclusions 649

References 649

24 Pathogenic Bacterial Sensors Based on Carbohydrates as Sensing Elements Haiying Liu 1 Introduction 660

2 Bacterial Surface Lectins 661

3 Surface Carbohydrate Structures of Pathogenic Bacteria 664

4 Carbohydrate Microarrays for Detection of Bacteria 668

5 Lectin Microarrays for Detection of Bacteria 670

6 Conjugated Fluorescent Glycopolymers for Detection of Bacteria 672

7 Glyconanoparticles for Detection of Bacteria 676

8 Carbohydrate-Functionalized Carbon Nanotubes for Detection of Bacteria 678

9 Conclusions and Future Perspectives 680

References 681

25 Aptamers and Their Potential as Recognition Elements for the Detection of Bacteria Casey C Fowler, Naveen K Navani, Eric D Brown and Yingfu Li 1 Functional Nucleic Acids 689

1.1 Properties of Nucleic Acids 690

1.2 Synthesizing, Sequencing and Modifying Nucleic Acids 692

1.2.1 DNA Polymerase and Polymerase Chain Reaction 692

1.2.2 RNA Polymerase and In Vitro Transcription 692

1.2.3 Reverse Transcription 693

1.2.4 Other Modifications 693

2 Isolation of Functional Nucleic Acids 694

2.1 Introduction to SELEX 694

2.2 Selection Methods 694

2.2.1 Bead and Column Based Selections 696

2.2.2 Polyacrylamide Gel Electrophoresis (PAGE) Based Selections 696

2.2.3 Capillary Electrophoresis (CE) Based Selections 697

2.3 Optimizing Functional Nucleic Acids 697

3 Aptamers: Properties and Targets 697

3.1 The Growing Aptamer Catalogue 698

3.2 Aptamer Specificity 698

3.3 Aptamer–Ligand Interactions 700

3.4 Aptamers vs Other Recognition Elements 700

4 Applications of Aptamers 701

4.1 Aptamers for Purification 701

4.2 Aptamers with Therapeutic Potential 702

4.3 Aptamers as Sensing Elements 702

4.3.1 Conformation-Dependent Fluorescent Sensors 703

4.3.2 Quantum Dot Sensors 703

4.3.3 Target Detection by Fluorescence Anisotropy 704

4.3.4 Enzyme Linked Aptamer Assays 705

4.3.5 Acoustic Sensors 705

4.3.6 Electrochemical Sensors 706

5 Aptamers for Detection of Pathogenic Bacteria 706

5.1 Categories of Microbial Agents to be Detected 707

5.1.1 Gram-Positive Bacteria 707

5.1.2 Gram-Negative Bacteria 708

5.2 Traditional Pathogen Detection Methods 708

5.3 Aptamers in Pathogen Detection 709

6 Conclusions 710

References 710

26 Protein Microarray Technologies for Detection and Identification of Bacterial and Protein Analytes Christer Wingren and Carl AK Borrebaeck 1 Introduction 715

1.1 Definition and Classification of Protein Microarrays 716

1.2 Functional Protein Microarrays 716

1.3 Affinity Protein Microarrays 719

1.4 Alternative Microarray Setups 720

2 Detection of Bacteria and Bacterial Protein Analytes 721

2.1 Serotyping of Bacteria 721

2.2 Detection of Pathogenic Organisms 721

2.3 Detection of Multiple Toxins 722

2.4 Simultaneous Detection and Identification of Bacterial Proteins and Bacteria 723

3 Detection of Diagnostic Markers, Toxin Regulators and Associated Protein Expression Profiles 724 3.1 Identification of Potential Diagnostic Markers and/or Vaccine Candidates 724

3.2 Disease State Differentiation and Identification of Diagnostic Markers 724

3.3 Identification of Potential Toxin Modulators/Regulators 725

3.4 Screening of Protein Expression Signatures Associated with Bacterial Infection 726

4 Conclusions and Future Perspectives 726

References 726

27 Bacteriophage: Powerful Tools for the Detection of Bacterial Pathogens Mathias Schmelcher and Martin J Loessner 1 Introduction 731

2 Detection by Phage Amplification 732

3 Detection Through Phage-Mediated Cell Lysis 734

3.1 Measurement of ATP Release 735

3.2 Detection of Other Cytoplasmic Markers 736

3.3 Measurement of Impedance 737

4 Detection Through Cell Wall Recognition, Phage Adsorption and DNA Injection 738

4.1 Immobilized Phage 738

4.2 Detection Through Phage-Encoded Affinity Molecules 738

4.3 Fluorescently Labeled Phage 740

5 Detection by Reporter Phage 741

5.1 Luciferase Reporter Phage (LRP) 743

5.2 Fluorescent Protein Reporter Phage 745

5.3 Other Reporter Phages 746

6 Other Detection Methods Using Phage 747

6.1 Phage Display for Production of Highly Specific Binding Molecules 747

6.2 Dual Phage Technology 749

7 Conclusions and Future Perspectives 750

References 750

28 Phage Display Methods for Detection of Bacterial Pathogens Paul A Gulig, Julio L Martin, Harald G Messer, Beverly L Deffense and Crystal J Harpley 1 Introduction 756

1.1 Why Detect Bacteria and What Tools Are Available? 756

1.2 Immunological Tools 756

1.3 Nucleic Acid-Based Tools 758

2 What Types of Antigen Detection Methods Are Being Developed? 758

3 Phage Display 759

3.1 Phage M13 760

3.2 Principles of Phage Display 760

3.3 Phages Versus Phagemids 762

3.4 Phage Display Formats 764

3.4.1 Random Peptides 764

3.4.2 Antibody Fragments 764

3.5 The Phages Themselves Are Not the Ultimate Tool 767

3.6 Using Phage Display 767

4 Review of Literature on Phage Display Against Bacterial Pathogens 769

4.1 Random Peptide Phage Display 770

4.2 scFv Libraries 772

4.3 Single Domain Antibodies (sdAbs) and Domain Antibodies (dAbs) 775

5 Summary of Our Results Using and Developing Phage Display scFv and Peptides 775

5.1 Panning Methods 776

5.2 Screening Methods 777

5.3 Genetic Modification of Phagemid Clones 777

5.4 Random Peptide Phage Libraries 777

6 New Directions 778

6.1 Proteins Based on Phage Display 778

6.1.1 Affibodies 778

6.1.2 Anticalins 778

6.1.3 Ankyrins 778

6.1.4 Trinectins 779

6.2 Alternatives to Phage Display 779

6.2.1 Aptamers 779

6.2.2 Ribosome Display 779

6.2.3 mRNA Display 780

7 Conclusions 780

References 780

29 Molecular Imprinted Polymers for Biorecognition of Bioagents Keith Warriner, Edward P.C Lai, Azadeh Namvar, Daniel M Hawkins and Subrayal M Reddy 1 Introduction 785

2 Principles of Molecular Imprinting 786

2.1 Imprinting Considerations 787

2.1.1 Versatility 787

2.1.2 Template Molecule 788

2.1.3 Functional Monomer 788

2.1.4 Cross-Linking 789

2.1.5 Polymerization 790

2.1.6 Solvent 790

2.2 Aqueous Phase MIP 791

2.2.1 Hydrogels 792

2.2.2 MIP Within Hydrogels 793

2.2.3 Polyacrylamide Gels—HydroMIPs 793

3 Solid Phase Extraction Based on MIPs for Concentrating Bioagents 795

3.1 Antibiotics 795

3.2 Mycotoxins 798

3.3 Nano-Sized Structures 799

3.4 Peptides and Proteins 800

3.5 Viruses 801

3.6 Bacterial Cells and Endospores 802

4 Biosensors Based on MIPs 803

4.1 MIP-based Sensors for Detection of Amino Acids 804

4.2 Molecular-Imprinted Films for Toxins 805

4.3 Microbial Imprinted Polymers 806

5 Conclusions and Future Perspectives 808

References 809

Part IV Microsystems 30 Microfluidics-Based Lysis of Bacteria and Spores for Detection and Analysis Ning Bao and Chang Lu 1 Introduction 817

2 Bench Scale Methods for Bacteria/Spore Lysis 818

3 Bacteria/Spore Lysis Based on Microfluidic Systems 820

3.1 Mechanical Lysis 8203.2 Chemical Lysis 8213.3 Thermal Lysis 8233.4 Laser-Based Lysis 8263.5 Electrical Lysis 827

4 Conclusions and Future Perspectives 829References 829

31 Detection of Pathogens by On-Chip PCR

Pierre-Alain Auroux

1 Introduction 833

2 Microfluidics 8342.1 History of Miniaturized Total Analysis System (TAS) 8342.2 Advantages of Miniaturized Analysis Systems 834

3 DNA Amplification 8353.1 A Brief History of DNA 8353.2 PCR Characteristics and Applications 8363.3 Components to Perform PCR 8373.4 PCR Process 8383.5 Conventional PCR 8393.6 Real-Time PCR: Apparatus and Detection Techniques 8403.7 On-Chip PCR 8413.7.1 Capillary-Based Thermocyclers 8423.7.2 Microdevice-Based Thermocyclers 8433.7.3 Static-Sample Systems 8433.7.4 Dynamic-Sample Systems 844

4 Minireview 846

5 Conclusions 848References 849

32 Micro- and Nanopatterning for Bacteria- and Virus-Based

Biosensing Applications

David Morrison, Kahp Y Suh and Ali Khademhosseini

1 Introduction 855

2 Fundamentals of Bacterial and Viral Surface Interactions 857

3 Technologies for Patterning 8583.1 Overview 8583.2 Photolithography 8583.3 Micromolding (Soft Lithography) 8593.3.1 Replica Molding 8593.3.2 Microcontact Printing 8593.3.3 Microtransfer Molding 8603.3.4 Capillary Force Lithography 8603.4 Scanning Probe Lithography 861

4 Biosensing Applications and Examples 8624.1 Overview 8624.2 Healthcare Applications 8644.3 Detection of Toxins in the Environment 8654.4 Real Devices and Challenges 866

5 Conclusions and Future Perspectives 866References 867

33 Microfabricated Flow Cytometers for Bacterial Detection

Sung-Yi Yang and Gwo-Bin Lee

1 Introduction 8691.1 Bio-MEMS 8711.2 Review of Microfabrication Techniques 8721.2.1 Bulk Micromachining Technique 8721.2.2 Surface Micromachining Technique 8721.2.3 LIGA 8721.2.4 Polymer-Based Micromachining Techniques for Microfluidic Devices 873

2 Operation Principles 8742.1 Cell Transportation and Focusing 8752.1.1 Hydrodynamic Approach 8752.1.2 Pneumatic Approach 8782.1.3 Electrokinetic Approach 8792.2 Cell Detection 8802.2.1 Optical Waveguide Approach 8812.2.2 Buried Optical Fiber Approach 8822.2.3 Large-Scale Optical System Approach 8822.3 Cell Sorting 8832.3.1 Hydrodynamic Sorting 8832.3.2 Pneumatic Sorting 8842.3.3 Electrokinetic Sorting 8852.3.4 Magnetic Sorting 885

3 Applications 8853.1 Environmental Monitoring 8863.2 Rapid Assessment of Bacterial Viability 8883.3 Rapid Analysis of Bacteria Levels in Food 8883.4 Antibiotic Susceptibility Testing 8893.5 Bacterial Detection in Blood and Urine 889

4 Conclusions and Future Perspectives 889References 890

34 Bacterial Concentration, Separation and Analysis by Dielectrophoresis

Michael Pycraft Hughes and Kai Friedrich Hoettges

1 Introduction 895

2 Theory 897

3 Applications of Electrokinetics to Bacteria 901

4 Toward an Integrated Detection System 904

5 Conclusions and Future Perspectives 905References 906

35 Ultrasonic Microsystems for Bacterial Cell Manipulation

Martyn Hill and Nicholas R Harris

1 Introduction 9091.1 Ultrasound and Bacterial Cells 9101.1.1 Cell Viability 9101.2 Ultrasound and Microfluidics 910

2 Relevant Ultrasonic Phenomena 9102.1 Axial Radiation Forces 9102.2 Lateral and Secondary Radiation Forces 912

2.3 Acoustic Streaming 9132.4 Cavitation 914

3 Applications of Ultrasonic Particle Manipulation 9143.1 Practical Considerations 9143.1.1 Transduction 9143.1.2 Mechanical Effects 9153.1.3 Construction 9163.2 Filtration and Fractionation of Cells 9173.2.1 Filtration and Concentration 9173.2.2 Fractionation of Cells 9203.2.3 Trapping of Cells 9213.3 Biosensor Enhancement by Forcing Cells to a Surface 922

4 Conclusions and Future Perspectives 924References 924

36 Recent Advances in Real-Time Mass Spectrometry

Detection of Bacteria

Arjan L van Wuijckhuijse and Ben L.M van Baar

1 Introduction 9291.1 General 9291.2 Scope 9301.3 MS in the Whole Cell Analysis of Bacteria 9301.3.1 The Definition of ‘Identity’ of Bacteria 9301.3.2 Mass Spectrometry of Bacteria 9311.4 Aerosol MS 9361.4.1 MS of Deposited Aerosols 9361.4.2 Direct MS of aerosols 938

2 Current State of the Technology 9392.1 Considerations on Aerosol MS of Bacteria 9392.2 Deposition and PyMS Based Technology 9402.3 Deposition and MALDI MS Based Technology 9412.4 Single Particle LDI MS Technology 9412.5 Single Particle MALDI MS Technology 943

3 Conclusions and Future Perspectives 946References 947Index 955

Evangelyn Alocilja

Biosystems and Agricultural Engineering

Michigan State University

East Lansing, Michigan

National Institute for Standards and Technology

EEEL, Semiconductor Electronics Division

Gaithersburg, Maryland

USA

Ben L M van Baar

TNO Defence

Security and Safety

Rijswijk, The Netherlands

Molecular Food Microbiology Laboratory

Department of Food Science

Purdue University

West Lafayette, Indiana

USA

Ning Bao

Department of Agricultural and Biological Engineering

School of Chemical Engineering

Birck Nanotechnology Center

Bindley Bioscience Center

Purdue University

West Lafayette, Indiana

USA

Arun K Bhunia

Molecular Food Microbiology Laboratory

Department of Food Science

Levente Bodrossy

Department of Bioresources Austrian Research Centres Seibersdorf, Austria

Carl AK Borrebaeck

Department of Immunotechnology and

CREATE Health Lund University Lund, Sweden

Eric D Brown

Department of Biochemistry and Biomedical Sciences and

Department of Chemistry McMaster University Hamilton, Canada

Daniel P Campbell

Georgia Tech Research Institute Atlanta, Georgia

USA

Fco Javier Del Campo

Instituto de Biotecnología y Biomedicina Departamento de Microbiología y Genética Universidad Autónoma de Barcelona Barcelona, Spain

S Yanyun Chen

Department of Food Service Purdue University West Lafayette, Indiana USA

Irina Ciubotaru

QuarTek Corporation Greensboro, North Carolina USA

xxvii

Department of Molecular Genetics and Microbiology

University of Florida College of Medicine

Genomic Research Laboratory

Division of Infectious Diseases

University of Geneva Hospitals

Geneva, Switzerland

Barry Glynn

The National Diagnostics Centre

National University of Ireland

Department of Molecular Genetics and Microbiology

University of Florida College of Medicine

Daniel M Hawkins

University of Surrey School of Biomedical and Molecular Sciences Guildford, Surrey

UK

Ryan B Hayman

Department of Chemistry Tufts University Medford, MA USA

Martyn Hill

School of Engineering Sciences The University of Southampton Southampton

UK

Kai Friedrich Hoettges

Centre for Biomedical Engineering University of Surrey

Guildford Surrey, UK

Jiˇri Homola

Institute of Photonics and Electronics Academy of Sciences

Prague Czech Republic and

Department of Chemical Engineering University of Washington

Seattle, Washington USA

Robert Horvath

Nanotechnology Centre

Cranfield University

Bedfordshire, UK

Michael Pycraft Hughes

Centre for Biomedical Engineering

National Institute of Standards and Technology

Chemical Sciences and Technology Laboratory

Biochemical Science Division

Harvard Medical School

Harvard-MIT Division of Health Sciences

Gwo-Bin Lee

Department of Engineering Science National Cheng Kung University Tainan, Taiwan

Yingfu Li

Department of Biochemistry and Biomedical Sciences and

Department of Chemistry McMaster University Hamilton, Canada

Haiying Liu

Department of Chemistry Michigan Technological University Houghton, Michigan

USA

Martin J Loessner

Institute for Food Science and Nutrition Zurich, Switzerland

Tracey Elizabeth Love

Defence Science and Technology Laboratory

Porton Down, Wiltshire UK

Chang Lu

Department of Agricultural and Biological Engineering School of Chemical Engineering Birck Nanotechnology Center Bindley Bioscience Center Purdue University West Lafayette, Indiana USA

Charles R Mace

University of Rochester Rochester, New York USA

Akira Matsumoto

Department of Bioengineering

Graduate School of Engineering

The University of Tokyo

Tokyo, Japan

Martin B McDonnell

Defence Science and Technology Laboratory

Porton Down, Wiltshire

Department of Materials Engineering

Graduate School of Engineering

The University of Tokyo

Biosystems and Agricultural Engineering

Michigan State University

East Lansing, Michigan

Francesc Xavier Muñoz

Centro Nacional de Microelectronica IMB-CNM-CSIC

Esfera UAB Campus Universidad Autónoma de Barcelona Barcelona, Spain

Raj Mutharasan

Department of Chemical and Biological Engineering Drexel University

Philadelphia, Pennsylvania USA

Azadeh Namvar

Department of Food Science University of Guelph Guelph, Ontario Canada

Naveen K Navani

Department of Biochemistry and Biomedical Sciences and

Department of Chemistry McMaster University Hamilton, Canada

Eric Olsen

Clinical Investigation Facility David Grant USAF Medical Center Travis Air Force Base, CA USA

Keat Ghee Ong

Department of Biomedical Engineering Michigan Technological University Houghton, Michigan

USA

Ilaria Palchetti

Dipartimento di Chimica Università di Firenze Sesto Fiorentino, Italy

Lynda Perry

Department of Food Service Purdue University West Lafayette, Indiana USA

Adrian Ponce

California Institute of Technology Jet Propulsion Laboratory Pasadena, California USA

Department of Materials Engineering

Graduate School of Engineering

The University of Tokyo

Service of Infectious Diseases

University Hospital of Geneva

Department of Internal Medicine

Ronald Turco

Department of Food Service Purdue University West Lafayette, Indiana USA

Anthony Turner

Cranfield University Bedfordshire UK

Arnold Vainrub

Department of Anatomy, Physiology, and Pharmacology

Auburn University Auburn, Alabama USA

Vitaly Vodyanoy

Department of Anatomy, Physiology, and Pharmacology

Auburn University Auburn, Alabama USA

Keith Warriner

Department of Food Science University of Guelph Guelph, Ontario Canada

Christer Wingren

Department of Immunotechnology and

CREATE Health Lund University Lund, Sweden

Arjan L van Wuijckhuijse

TNO Defence Security and Safety Rijswijk, The Netherlands

Sung-Yi Yang

Department of Engineering Science National Cheng Kung University Tainan, Taiwan

Ahmed E Yousef

Professor of Food Microbiology

Department of Food Science and Technology

and

Department of Microbiology

Parker Food Science Building

Ohio State University

Introduction

Introduction to Pathogenic Bacteria

Tracey Elizabeth Love and Barbara Jones

Abstract

This chapter is a brief introduction to pathogenic microorganisms and also discusses virulence factors

An understanding of virulence factors is important, as they represent potential targets for the detection ofmicrobial pathogens Sources and routes of infection are also briefly discussed with reference to specificexamples There are a number of ways in which infection could be acquired, including via contaminatedfood and water; hospital acquired infection; “naturally acquired” infection; and intentional infection, forexample, through the use of biological warfare agents The focus of the review is predominantly on humanpathogens However, there are a range of other microbial pathogens of particular importance in otherareas; for example, animal and plant pathogens, which will not be discussed Finally, a brief overview ofthe detection of pathogenic bacteria is presented

1 Pathogenic Microorganisms

Over many years there has been considerable debate as to the exact definitions of pathogenicityand virulence These two words are often used interchangeably, but pathogenicity has beendefined as the ability of an organism to cause disease and virulence as the relative severity of thedisease caused by the organism (Watson and Brandly 1949) It has become increasingly apparentthat virulence is highly complex and is dependent on the interaction between the host and themicroorganism (Casadevall and Pirofski 2001) Taking into account the problems associatedwith defining virulence, virulence factors have also been difficult to characterise Two defini-tions that have been put forward are that a virulence factor is (1) a “component of a pathogenthat when deleted specifically impairs virulence but not viability” (Wood and Davis 1980); or(2) a “microbial product that permits a pathogen to cause disease” (Smith 1977) However,these often do not apply to infections caused by commensal or opportunistic pathogens, whereoften classic virulence determinants do not exist Furthermore, the definitions may not accountfor host tissue damage that has been caused by the induction of a particular part of the host’simmune response, such as cytokine synthesis (Henderson et al 1996) Therefore an under-standing of virulence factors is important, as these can often be used to specifically detectpathogenic microorganisms Classical virulence factors include factors that aid in a number ofstages of infection:

1) host cell attachment;

2) entry to the host cell;

Tracey Elizabeth Love • Defence Science and Technology Laboratory, Porton Down, Wiltshire, UK.

Barbara Jones • National Institute of Standards and Technology, Chemical Sciences and Technology Laboratory,

Biochemical Science Division, Gaithersburg, Maryland, USA.

M Zourob et al (eds.), Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems,

© Springer Science+Business Media, LLC 2008

3

3) evasion of detection by the hosts immune system;

4) intracellular or extracellular replication and inhibition of phagocytosis

Virulence factors can be either requisite, where the gene products discriminate betweenpathogenic and nonpathogenic species; or contributory factors, that alter the severity of thedisease Again, the ability to cause disease and its severity will also be dependent on the immunestatus of the host Contributory virulence factors do not fulfil the definition of virulence factors;nor do they separate pathogenic from nonpathogenic species, as they may be found in a widerange of microorganisms but still have a role in damage to host cells A general consensus ofopinion has been that regardless of the function of a gene product, if its expression leads todamage of the host cell then it is a virulence factor Therefore Casadevall and Pirofski (1999)suggest that virulence factors should be defined as “attributes that mediate host damage.”Bacterial pathogens usually possess a number of virulence factors that are essential in estab-lishing infection and causing disease Classical virulence factors include toxins, as well asmolecules that are involved in adherence, invasion of the host, evasion of the host’s immuneresponse, and iron acquisition

1.1 Toxins

Some microorganisms (e.g., Bacillus anthracis) produce toxins that are the major cause of

clinical symptoms observed in patients Toxins can be integral parts of the bacterium, such aslipopolysaccharide (endotoxins), or secreted molecules (exotoxins) Toxins often perform otherfunctions, such as the making of adhesins (Tuomanen and Weiss 1985) Toxin secretion may also

be regulated as part of an orchestrated response by the bacterium The lipopolysaccharide (LPS)content of pathogenic Gram-negative cell walls is contained within a microorganism and usuallyreleased when the cell dies or is broken down (by autolysis or by the host’s immune response).Unlike exotoxins, endotoxins are believed not to have any direct enzymatic action; and it

is the lipid A portion, usually embedded within the bacterial membrane, that is believed to bethe toxic component As LPS is released from the bacterial cell, a number of host moleculesinvolved in the inflammatory response are released (e.g., cytokines) One of the most importantcytokines released is tumour necrosis factor- (TNF-) This molecule usually prevents thespread of a localised infection However, the rapid stimulation of high levels of TNF- withinthe bloodstream results in fever, damage to host tissue, an alteration of metabolism, and theproduction of further cytokines (IL-6, IL-8, IL-1, and PAF, platelet activating factor) Thesecytokines produce further damage to host cells and tissue resulting in a dramatic decrease

in blood pressure and reduced blood flow to major organs leading to multiple organ failure(Tracey and Cerami 1993, Rink and Kirchner 1996) Exotoxins can be divided into a number

of broad categories summarised below

Fimbrial adhesins or pili can be observed by electron microscopy as hair-like structuresthat are present predominantly on the surface of Gram-negative bacteria (nearly all Gram-positive organisms do not possess pili) A number of Gram-negative pathogens utilise pili for

adherence such as Vibrio cholerae and Neisseria gonorrhoeae Originally it was suggested

that pili were homopolymeric, composed of a number of repeating pilin (or fimbrin) subunits.However, it has become apparent that for many pili (e.g., Type I and Pap pili) these structuresare heteropolymeric Furthermore, the protein subunits located either at the tip or the base ofthese structures are important for a particular function For example, the minor protein subunitslocated at the distal end of the pilus (the tip fibrillum) are frequently involved in attachment

to the host cell receptor (Hultgren et al 1993) Pili have the capacity to attach to a number

of different receptors (Table 1.1) and genetic variation in the tip adhesin confers differences

in binding affinity to host cell receptors, which allows for differences in the tissue tropism

Bacterial DNA may encode multiple operons for fimbrial expression; for example, Salmonella.

typhimurium encodes four fimbrial operons ( fim, lpf, pef, and agf ) Deletion of individual

fimbrial operons resulted in a minor reduction in virulence, but a quadruple mutant demonstratedsignificant attenuation compared to the wild type strain (Van der Velde et al 1998) Thissuggests that deletion of a single component of virulence can be compensated by the presence

of related virulence factors in the case of some microorganisms

Curli are another form of pilus type adhesins found in some strains of Escherichia coli and

Salmonella entereditis spp They are highly stable, thin, irregular surface structures that facilitate

binding to host proteins such as plasminogen and fibronectin All pili are assembled in a highlyordered manner and although the assembly mechanism may vary, common characteristics areoften observed (Soto and Hultgren 1999) Nonpilus adhesins can be found in Gram-negative,Gram-positive, and mycobacterial pathogens Examples of nonfimbrial adhesins are summarised

in Table 1.2

Table 1.1. Examples of bacterial toxins and modes of action (compiled from Merrit and Hol 1995,Schiavo et al 1992, Welch 1991, Savarino et al 1993, Falzano et al 1993, and Schmitt et al 1999)

Type of Toxin Mode of Action Example Organisms

A-B Toxins (Type

III toxins)

The A subunit has enzymatic activity that mediates toxicity The B-subunit binds to the host cell receptor and allows for delivery of the A-subunit.

Vibrio cholerae (cholera toxin), Bordetella pertussis (pertussis

Host Cell Function

One group affects the host cell cytoskeleton by modification of Rho family (small GTP binding proteins) resulting in various detrimental effects

on actin polymerisation (toxin dependent).

E coli (CNF, cytotoxic necrotising

factor), Clostridium difficile

Neisseria meningitidis, Haemophilus influenzae

Heat Stable Toxins Heat stable toxins – binding of the toxin to its

receptor stimulates activation of guanylate cyclase increasing intracellular GMP in turn causing dramatic ion flux changes.

Enterotoxinogenic E coli, Yersinia

enterocolitica, Vibrio cholerae.

Superantigens

(Type I Toxins)

Immunostimulatory toxins that bind to MHC class

II molecules stimulating the production of T-cells and triggering the release of cytokines involved in the inflammatory response This causes fever, shock, and erythematous rash.

Staphlococcus aureus, Streptococcus pyogenes, Staphylococcal enterotoxin.

Table 1.2. Examples of Gram negative pilin adhesins (modified

from Wizemann et al 1999)

Adhesin Strain Host Receptor

PapG Klebsiella pneumoniae Gal (1–4)Gal

MrkD Escherichia coli Type V collagen HifE Haemophilus influenzae Sialylyganglioside-GM1

Bacteria can also produce other molecules that are involved in host cell attachment,such as intimins These allow for close associations between the pathogens and result inrearrangement of the cytoskeleton, interference with host cell signalling, and possibly in bacterialinternalisation A more novel mechanism of host cell attachment is a pathogen secreted receptor,which is endocytosed by the host cell and subsequently presented on the host cell surface in

a phosphorylated form This then functions as a receptor for the bacterial cell, for example in

enteropathogenic E coli (Kenny et al 1997).

Other surface structures may also be involved in specific and nonspecific adherence to ahost cell, such as a slime layer, capsule, LPS, techoic acid and lipotechoic acid For example, the

capsular glucose and mannan polysaccharides of Mycobacteria species adhere to complement

receptor 3 and the mannose receptors of host cells (Daffe and Etienne 1999) The techoic acids

of Staphylococcus and Streptococcus spp can also function as adhesins (Walker 1998) Slime

layers and capsules are usually composed of polysaccharides, but can be made of polypeptides

If the surrounding material is unorganised and loosely attached to the cell wall it is referred to

as a slime layer, whereas an organised layer that is firmly attached to the bacterial cell is termed

a capsule Both may mediate specific or nonspecific attachment, but do not necessarily have

a role in pathogenicity or virulence Many adhesins have also demonstrated important roles inevasion of the host immune system (e.g., capsules, LPS, and techoic and liptotechoic acids).Both Gram-negative and Gram-positive pathogens often express and utilise a large reper-toire of adhesins (Tables 1.1 and 1.2) Adhesion can occur as a protein-protein or protein-carbohydrate interaction, and a vast array of host molecules are used as adhesin targets(Table 1.3) For example, surface immunoglobulin, glyocproteins, glycolipids, and extracellularmatrix proteins such as fibronectin, collagen, or laminin have been shown to interact withadhesins (Finlay and Falkow 1997)

Table 1.3. Examples of afrimbrial adhesins in Gram-negativeand Gram-positive bacteria (Brubaker 1995)

Pertactin Bordetella pertussis Integrin

HMW1/HMW2 Haemophilus influenzae Human epithelial cells

Envelope antigen F1 Yersinia pestis Not known

Le b binding adhesion Helicobacter pylori Fucosylated Le b

histocompatibility blood group antigens CpbA/SpsA/PbcA/PspC Streptococcus pneumoniae Cytokine activated

epithelial and endothelial cells.

P1, Pac Streptococcus mutans Salivary glycoprotein

FnbA, FnbB Staphyloccus aureus Fibronetin

1.3 Invasion

As discussed previously, intimins often mediate a close association with the host cell,which can lead to internalisation of the pathogenic microorganism Classes of adhesin thatfacilitate entry into the host cell are termed invasins and are the most common mode of entry of

a bacterial pathogen into both phagocytic and nonphagocytic cells (Finlay and Falkow 1997)

In phagocytic cells, pseudopod formation occurs as cell signals mediate cytoskeletal ments, resulting in polymerisation and depolymerisation of actin Internalisation of a bacterialpathogen by cells other than professional phagocytes is mediated by invasin-activated actinrearrangements This has the same effect as in a phagocytic cell, resulting in forced phago-cytosis of the pathogen (Bliska et al 1993, Rosenshine and Finlay 1993) Some pathogenicspecies also utilise host microtubules (polymerised tubulin) to enter nonphagoctyic cells e.g.,

rearrange-N gonorrrhoae or K pneumoniae The exact mechanism is undefined, but they do not appear to

utilise this mechanism as an essential virulence factor Some pathogens target phagocytes andmay use phagocytic pathways for internalisation Once the pathogen is internalised, it residesinitially within a membrane vesicle The pathogen can either remain within or escape fromthe vesicle Many pathogenic species remain within the vesicle and have evolved mechanisms

to evade the cellular response of the host For example, capsules and LPS can serve as aprotective barrier for internalised pathogens Other factors, such as the secretion of enzymesthat neutralise oxygen radicals and proteolytic enzymes that can degrade host cell lysosyme,are also important for intracellular survival Exploitation of host cell signals may also occur(e.g., acidic pH) to activate replication and the initiation of the expression of other virulencefactors or cascades required for intracellular survival Pathogenic bacteria may also have theability to replicate within the host cell and spread to other host cells

1.4 Evasion of the Host Immune Response

Many surface elements of bacterial pathogens serve to aid in the evasion of the host’simmune response Capsules consisting of a mix of polysaccharide, protein, and glycoproteinprevent complement activation by inhibition of the assembly of C3 convertase on the bacterialcell surface using a variety of mechanisms Prevention of C3 convertase assembly on thebacterial surface inhibits the efficiency of phagocytosis, as opsonisation of the pathogen isless likely to occur C3 convertase may assemble beneath the capsule, and the C3b moleculemay also be able to diffuse through; however, the capsular network blocks subsequent contactwith phagocytic receptors (Taylor and Roberts 2005) Lack of C3 convertase on the surfacealso reduces the probability of the formation of a membrane attack complex (MAC) on theunderlying bacterial surface The capsule itself may provoke an immune response One way bywhich a number of pathogenic species have overcome this is to produce a nonimmunogeniccapsule composed of polysaccharides similar to host polysaccharides—for example, sialic orhyaluronic acid LPS also aids in the evasion of complement activation and phagocytosis TheLPS O-antigen blocks C3 convertase assembly through the binding of sialic acid Variation

in the length of the LPS O-antigen side chain prevents the assembly of an effective MACconferring serum resistance, which is important for establishing systemic infections Anotherexample of evasion of complement activation and phagocytosis is the production of enzymesand toxins to prevent the migration of phagocytes to the infected site (for example, through theenzymatic degradation of a the chemoattractant C5a) (Taylor and Roberts 2005)

Toxins may also protect pathogens against phagocytosis by killing phagocytes andreducing the production of toxic reactive oxygen intermediates (oxidative burst) Many highlyvirulent pathogenic bacteria target phagocyte receptors and also have the ability to survivephagocytosis by polymorphonuclear leukocytes (PMNs), macrophages, and monocytes Manystrategies exist for surviving within these cells, such as:

1) prevention of phagosome-lysosome fusion;

2) release from the phagosome prior to lysosome fusion;

3) secretion of molecules that reduce the toxic effects of the components of the lysosomeinto the phagolysosome (e.g., the expression of enzymes such as superoxide dismutase thatdetoxify oxygen radicals)

Other mechanisms include the production of cell walls that are resistant to degradation bylysosyme, and interference with signalling pathways through the production of enzymes thatare homologous to host cell enzymes

Pathogens can also evade the host’s immune response by inhibiting the production ofantibodies by utilising a range of strategies including phase variation (constant switching ofthe expression of surface antigens) Use of this strategy allows the microorganism to avoidantibodies, as those produced will only be effective against the forms previously expressed(Meyer 1991) Strain variation of immunodominant and highly expressed proteins may confer

a selective advantage to pathogenic species Furthermore, surface components such as LPS,capsules, S-layers, flagella, and outer membrane proteins may demonstrate antigenic variation(Brunham et al 1993) As mentioned previously, nonimmunogenic structures or layers such

as capsular polysaccharides or carbohydrates may be produced that resemble those of the hostand even mimic the function of host cell proteins (Stebbins and Galán 2001) Bacteria mayalso be coated in host cell proteins, such as fibronectin and collagen The coating of bacteriawith host antibodies can prevent opsonisation, possibly through prevention of recognition byspecific antibodies to surface located antigens or the inhibition of complement assembly One

example of this is protein A of S aureus and protein G of S pyogenes, which bind to the Fc

portion of immunoglobulins, thus covering the pathogen in antibody Some pathogenic speciesalso express specific iron binding receptors Although the primary function of these receptors

is iron acquisition, it has also been suggested that they may have a role in masking surfaceantigens

1.5 Iron Acquisition

Iron is required for bacterial replication; however, low iron concentrations are oftenfound within a host, particularly within humans Therefore for survival and growth, someform of iron acquisition mechanism is required Siderophores are high affinity iron bindinglow molecular weight compounds, secreted by the bacteria to chelate iron (Neilands 1995).The iron-siderophore complex is then bound by siderophore receptors located on the bacterialsurface It has been demonstrated that this type of iron acquisition mechanism may contribute

to bacterial virulence, but this has not always been found to be the case In humans most iron

is bound to proteins such as ferratin, lactoferratin, hemin, or transferrin It has been shownthat some pathogens are able to utilise these as an iron source through binding to receptors

on the bacterial surface, although the exact mechanism of the removal of the iron has notbeen clearly defined Another potential method of iron acquisition is the production of othervirulence factors such as exotoxins, invasins, adhesins, and outer membrane proteins whereexpression is activated by low iron concentrations (Litwin and Calderwood 1993) It has beensuggested that exotoxins kill host cells, thus releasing iron stores which can then be utilised bythe pathogen Many bacteria have more than one method of iron acquisition, therefore deletionmay be compensated by another system

1.6 Regulation of Virulence Factors

Infection of a host with a pathogen presents the microorganism with adaptive changesrequired for survival and replication within different environments These changes may be in

response to a number of biochemical and physical parameters such as temperature, pH, ionconcentration, growth phase, osmolarity, oxygen, calcium, and iron levels (Gross 1993) Manyvirulence factors will be required only when an organism is within a host, others may also beessential for survival outside the host Once a pathogen is within a host, environmental signalswill induce the switching on and off of various virulence genes, dependent on the stage ofinfection It is common for a single regulatory element to control the expression of numerousvirulence factors that may not be related; these are sometimes termed global regulators It hasalso become apparent that virulence factor expression is a complex interdependent processrelying on various cues from the host and the pathogen The expression of a number ofvirulence factors may be coordinated simultaneously by several different regulatory elements;alternatively, a single virulence factor can come under the control of several regulatory elements.

2 Sources and Routes of Infection

2.1 Natural Infection

Pathogenic bacteria are widely distributed and can be found in the soil, other animals orhumans, food, or water, depending on bacterial species All these represent potential sources

of infection Infection can be via a number of routes, such as inhalation, ingestion, abrasion

to the skin, contaminated blood, or the bite of an insect vector The way in which infection isestablished will again depend on the microorganism of concern and may also have an effect

on the predicted outcome of the disease For example, an organism such as B anthracis, the

aetiological agent of anthrax, has a number of different forms In the case of humans, there arethree main forms of the anthrax disease: cutaneous, inhalational, and gastrointestinal Any ofthese types of infection can result in systemic anthrax, which is nearly always fatal (Mock andFouet 2001) The severity of disease is usually greatest with inhalational anthrax, and if untreatedthis form of the disease has a mortality rate approaching 100% (Turnbull 1991, Webb 2003).Cutaneous anthrax is often self-limiting with or without the appropriate treatment (Hambletonand Turnbull 1990) There are a number of diseases that are of importance throughout theworld; however, there are three areas for which the rapid sensitive detection of pathogenicmicroorganisms has been addressed in this review: food- and water-borne pathogens, hospitalacquired infections, and the intentional use of pathogenic bacteria for biological warfare orbioterrorism These are discussed mainly in the context of human infections, but obviouslythere is a range of pathogenic microorganisms that can infect animals and plants and are ofimportance, but that will not be discussed in detail within this review

2.2 Food and Water

There are many different types of food-borne pathogens such as bacteria, viruses,parasites, prions, and bacterial toxins The symptoms of illness usually comprise mild or severegastrointestinal discomfort such as nausea, vomiting, and/or diarrhoea, but can also extend tolife-threatening renal (kidney), hepatic (liver), and neurological complications Infection fromfood-borne pathogens are often unreported due to generic diagnoses such as “stomach flu” andthe fact that many individuals do not seek medical treatment unless symptoms become severe.The duration of illness varies and is typically short for bacterial infection, but can be chronicfor viral or parasitic infections Well known factors contributing to bacterial contamination offoods include the improper handling and storage of foods, inadequate cooking or reheating,cross-contamination between raw and cooked foods or fresh produce, and poor hygiene offood service workers Overall, food-borne diseases are estimated to cause more than 76 millionillnesses each year in the United States (Mead et al 1999) Common foodborne pathogens

identified by the FDA are Escherichia coli, Salmonella, Shigella, Campylobacter, Yersinia

enterocolitica and Yersinia pseudotuberculosis, Vibrio, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Clostridium botulinum, as well as yeasts,

moulds, and mycotoxins Food-borne pathogens differ widely in their incidence, infectiveness,and symptoms of disease Some food-borne infections are merely uncomfortable, while somecan become life-threatening

The issue of the presence of food-borne pathogens is exacerbated by the low threshold ofinfectious organisms required to cause severe illness For example, as few as 15 Salmonellosisorganisms can lead to a severe condition (US Food and Drug Administration 2007) Therefore,the choice of method for detection and analysis of the pathogens is paramount Two distinctgroups are generally reported: conventional methods and rapid detection methods Though manyrapid detection methods are used in the initial screening of suspected foods, confirmatory testingfor positive results using conventional methods is usually performed (Arora et al 2006) Conven-tional methods for detecting food-borne pathogens require selective enrichment of the pathogen,plating and characterization from colony and organism morphology, and traditional proceduressuch as sugar fermentation and immunoprecipitation These conventional methods are oftenapproved as a method of identification by regulatory bodies but are often labour intensiveand can require up to five days in the case of some culture methods However, they remainthe standard for food-borne pathogen detection and identification The term “rapid method”

is used to describe an array of tests including: polymerase chain reaction (PCR) and DNAhybridization, real-time polymerase chain reaction (RQ-PCR), nucleic acid-based sequenceamplification (NASBA), enzyme-linked immunosorbent assay (ELISA), and restriction enzymeanalysis (REA), among others

2.3 Hospital Acquired Infections

Hospital acquired infections occur throughout the world in both developed and veloped countries, are one of the most common causes of morbidity in patients (Ponce deLeon 1991), and have a significant economic impact (Ponce de Leon 1991, Plowman et al 1999,Wakefield et al 1988, Coella et al 1993, Wenzel 1995) Infections are usually in the lowerrespiratory tract, in surgical wounds or the urinary tract, and a WHO study indicated that thehighest frequency of infection was in intensive care and acute surgical and orthopaedic wards(WHO 2002) The patient population is often immunocompromised due to age, the presence ofanother disease, or immunosuppressive treatments such as chemotherapy, making individualsmuch more suspectible to infection, particularly with opportunistic pathogens (Geddes andEllis 1985) As in the case of food-borne pathogens, there are a number of organisms whichcan cause infection—including bacteria, viruses, fungi, and parasites An individual could beinfected by person-to-person transmission, by bacteria in their own flora, or by contaminatedobjects they have come into contact with The majority of hospital acquired infections caused

nonde-by bacteria are Staphylococcus aureus, coagulase-negative staphylococci, enterococci, or

enter-obacteriacae (WHO 2002) One other factor that has been highlighted in recent years is theemergence of bacteria that are resistant to many antimicrobial therapies, sometimes resulting

in multidrug-resistant strains or “super bugs.” One of the overriding reasons for this is thought

to be the widespread indiscriminate use of antibiotics to treat infections Bacteria that havedeveloped multidrug resistance include strains of staphylococci, pneumococci, enterococci, andtuberculosis (Longworth 2001)

2.4 Intentional Infection—Biological Warfare

Biological agents with potential use in biological warfare (BW) can broadly be dividedinto three categories: toxins (that have a range of sources such as animals, bacteria, and plants),

viruses, and bacteria The number of agents that could be utilised is extensive; however, of these

a significantly smaller proportion could be effectively disseminated through the aerosol route,considered the most likely route for a large-scale attack (Christopher et al 1997, Christopher

et al 1999, Eitzen 1997, Eitzen et al 1998, Franz 1997, Kortepeter and Parker 1999, Peters

and Dalrymple 1990) Microbial pathogens that could be used include: Bacillus anthracis,

Yersinia pestis, Venezuelan equine encephalitis (VEE), Francisella tularensis, Variola virus,

and the haemorrhagic fever viruses (arenaviruses, filoviruses, flaviviruses and bunyaviruses).Toxins also represent a threat from potential use as BW agents; examples include those that

could be isolated from Clostridium botulinum, Ricinus communis, trichothecane mycotoxins,

or staphylococcal enterotoxins (Hawley and Eitzen 2001) Some of these, such as VEE, are

considered incapacitating agents, and others, such as Bacillus anthracis, as lethal agents (Hawley

and Eitzen 2001) Of all the lethal agents the most serious to the human host are haemorrhagicfever viruses such as the Ebola virus, Lassa fever, or the Marburg virus, as no prophylaxis

or vaccines are currently available (Koch et al 2000) However, these viruses are much moredifficult to produce than toxins or bacteria Virus propagation requires more sophisticatedequipment and a higher degree of expertise such as tissue culture Bacteria by comparison aremuch easier to grow once a source of the pathogen has been found In the case of BW agents,often the LD50 or ID50 in humans is predicted to be low, and any potential use needs to beidentified quickly and at a relatively low level, making the requirement for rapid, specific, andsensitive detection of these pathogenic microorganisms essential

3 Detection of Pathogenic Microorganisms

The rapid, sensitive, and specific detection of pathogenic microorganisms is essential ifeffective treatment is to be provided to a susceptible population In the case of bacteriologicaltesting, traditional microbiology has proved a time consuming procedure Organisms have

to be isolated and grown, and usually a series of biochemical tests must be completed foridentification (Helrich 1990, Kaspar and Tartera 1990) Techniques such as the polymerasechain reaction (PCR) used for the amplification of pathogen-specific DNA sequences haveproved to be sensitive However, when using environmental samples, a degree of samplepreparation is required since impurities contained within the sample may inhibit the PCR.Furthermore, the use of small sample volumes (sometimes 1 l) means that the sample oftenhas to be concentrated to obtain the desired sensitivity (Radstrom et al 2004)

Biosensors are particularly attractive as a means to detect and identify potential pathogenicmicroorganisms due to their potential specificity and sensitivity (although this is also governed

by the choice of recognition element), together with the provision of information in near realtime Biosensors also allow the analysis of complex sample matrices (Hobson et al 1996,Ivnitski et al 1999) To provide protection, i.e., timely warning of the presence of a pathogen,environmental samples are often analysed using biosensors This presents an additional problem,

in that other microorganisms will also be present within the sample The detector needs to beable to discriminate the pathogen of interest from the background, and this can be achieved in

a number of ways These include (a) detection of an increase in the number of particles, (b)detection of an increase in biological particles, (c) detection of pathogenic biological agents,

or (d) the specific identification of a biological agent Specific detection is dependent on theinteraction of the target analyte (e.g., a protein) with a recognition element (e.g., an antibody).The use of biosensors for sensitive specific detection of a pathogenic microorganism stillremains a significant challenge, and success is often dictated by the nature of the detectionelement (the specific ligand) and the choice of target analyte (Labadie and Desnier 1992)