Width: 24mm

Width: 24mm

Edited byOmar A. Oyarzabal and

Sophia Kathariou

DNA Methods in Food

SafetyMolecular Typing

of Foodborne and Waterborne

Bacterial Pathogens

Molecular typing of foodborne pathogens has become an indispensable tool in epidemiological studies. Thanks to these techniques we now have a better understanding of the distribution and appearance of bacterial foodborne diseases and have a deeper knowledge of the type of food products associated with the major foodborne pathogens. Within the molecular techniques DNA-based techniques have prospered for more than 40 years and have been incorporated in the � rst surveillance systems to monitor bacterial foodborne pathogens in the United States and other countries. However DNA techniques vary widely, and many microbiology laboratory personnel working with food and/or water face the dilemma of which method to incorporate.

DNA Methods in Food Safety: Molecular Typing of Foodborne and Waterborne Bacterial Pathogens succinctly reviews more than 25 years of data on a variety of DNA typing techniques, summarizing the different mathematical models for analysis and interpretation of results, and detailing their ef� cacy in typing different foodborne and waterborne bacterial pathogens, such as Campylobacter, Clostridium perfringens, Listeria, Salmonella, among others. Section I describes the different DNA techniques used in the typing of bacterial foodborne pathogens, while Section II deals with the application of these techniques to type the most important bacterial foodborne pathogens. In Section II the emphasis is placed on the pathogen, and each chapter describes some of the most appropriate techniques for typing each bacterial pathogen.

The techniques presented in this book are the most signi� cant in the study of the molecular epidemiology of bacterial foodborne pathogens to date. It therefore provides a unique reference for students and professionals in the � eld of microbiology, food and water safety, and epidemiology and molecular epidemiology.

Dr Omar A. Oyarzabal is Vice President of Technical Services at IEH Laboratories and Consulting Group, Seattle, WA, USA.

Dr Sophia Kathariou is Professor of Bioprocessing and Nutrition Sciences at the Department of Food, North Carolina State University, Raleigh, NC, USA.

Also Available from Wiley BlackwellPractical Food Safety: Contemporary Issues and Future DirectionsRajeev Bhat (Editor), Vicente M. Gomez-Lopez (Editor) ISBN: 978-1-118-47460-0

Guide to Foodborne Pathogens, 2nd EditionRonald G. Labbé (Editor), Santos García (Editor) ISBN: 978-0-470-67142-9

www.wiley.com/wiley-blackwell

www.wiley.com/go/food

Also available as an e-book

DN

A M

ethods in Food S

afetyM

olecular Typing of Food

borne and

Waterb

orne Bacterial P

athogens

Oyarzab

al and

Kathariou

Thumbnail.jpg

ps0303File AttachmentThumbnail.JPG

DNA Methods in Food Safety

DNA Methods in Food SafetyMolecular Typing of Foodborne and Waterborne Bacterial Pathogens

Edited by

Omar A. OyarzabalVice President of Technical Services at IEH Laboratories and Consulting Group, Seattle, WA, USA

Sophia KathariouProfessor of Bioprocessing and Nutrition Sciences at the Department of Food, North Carolina State University, Raleigh, NC, USA

This edition first published 2014 © 2014 by John Wiley & Sons, Ltd

Registered OfficeJohn Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

Editorial Offices9600 Garsington Road, Oxford, OX4 2DQ, UKThe Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK111 River Street, Hoboken, NJ 07030-5774, USA

For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell.

The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher.

Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book.

Limit of Liability/Disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. If professional advice or other expert assistance is required, the services of a competent professional should be sought.

Library of Congress Cataloging-in-Publication Data

DNA methods in food safety : molecular typing of foodborne and waterborne bacterial pathogens / edited by Omar Oyarzabal, Sophia Kathariou. p. ; cm. Includes bibliographical references and index. ISBN 978-1-118-27867-3 (cloth)I. Oyarzabal, Omar A., editor. II. Kathariou, Sophia, editor. [DNLM: 1. Food Safety–methods. 2. DNA Fingerprinting–methods. 3. Food Microbiology–methods. 4. Molecular Typing–methods. 5. Water Microbiology. WA 695] RA601.5 363.19′26–dc23

2014013804

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Set in 10.5/13pt Times Ten by SPi Publisher Services, Pondicherry, India

1 2014

Contents

List of Contributors viiPreface xiii

Section I Typing Method, Analysis, and Applications 1

1 Polymerase Chain Reaction-Based Subtyping Methods 3Yi Chen and Insook Son

2 Pulsed-Field Gel Electrophoresis and the Molecular Epidemiology of Foodborne Pathogens 27Mohana Ray and David C. Schwartz

3 Multilocus Sequence Typing: An Adaptable Tool for Understanding the Global Epidemiology of Bacterial Pathogens 47Stephen J. Knabel

4 High-Throughput Sequencing 65Xiangyu Deng, Lee S. Katz, Patricia I. Fields, and Wei Zhang

5 Analysis of Typing Results 85João André Carriço and Mário Ramirez

6 Databases and Internet Applications 113G. Gopinath, K. Hari, R. Jain, M. H. Kothary, K. G. Jarvis, A. A. Franco, C. J. Grim, V. Sathyamoorthy, M. K. Mammel, A. R. Datta, B. A. McCardell, M. D. Solomotis, and Ben D. Tall

7 The Transformation of Disease Surveillance, Outbreak Detection, and Regulatory Response by Molecular Epidemiology 133David A. Sweat

vi Contents

Section II Pathogens 163

8 The Genus Bacillus 165Monika Ehling-Schulz and Ute Messelhäusser

9 Molecular Typing of Campylobacter jejuni 185Catherine D. Carrillo and Omar A. Oyarzabal

10 DNA Typing Methods for Members of the Cronobacter Genus 205Susan Joseph and Stephen Forsythe

11 Molecular Subtyping Approaches for Pathogenic Clostridium spp. Isolated from Foods 249Brian H. Raphael, Deborah F. Talkington, Carolina Lúquez, and Susan E. Maslanka

12 Molecular Characterization of Shiga Toxin-Producing Escherichia coli 275Pallavi Singh and Shannon D. Manning

13 Molecular Subtyping Methods for Listeria monocytogenes: Tools for Tracking and Control 303Sara Lomonaco and Daniele Nucera

14 Salmonella 337Aaron M. Lynne, Jing Han, and Steven L. Foley

15 Vibrio cholerae 359Dong Wook Kim

Index 381

List of Contributors

João André CarriçoInstituto de Microbiologia, Instituto de Medicina MolecularFaculty of Medicine, University of LisbonLisboa, Portugal

Catherine D. CarrilloCanadian Food Inspection AgencyOttawa Laboratory (Carling)Ottawa, Ontario, Canada

Yi ChenCenter for Food Safety and Applied NutritionFood and Drug AdministrationCollege Park, MD, USA

A. R. DattaOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Xiangyu DengCenter for Food SafetyUniversity of GeorgiaGriffin, GA, USA

Monika Ehling-SchulzInstitute of Functional MicrobiologyDepartment of Pathobiology University of Veterinary Medicine, VeterinaerplatzVienna, Austria

viii List of Contributors

Patricia I. FieldsEnteric Diseases Laboratory Branch, Division of Foodborne, Waterborne and Environmental DiseasesNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and PreventionAtlanta, GA, USA

Steven L. FoleyDivision of MicrobiologyFDA-National Center for Toxicological ResearchJefferson, AR, USA

Stephen ForsytheSchool of Science and TechnologyNottingham Trent UniversityNottingham, UK

A. A. FrancoOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

G. GopinathOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

C. J. GrimOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Jing HanDivision of MicrobiologyFDA-National Center for Toxicological ResearchJefferson, AR, USA

K. HaricBio, IncFremont, CA, USA

R. JaincBio, IncFremont, CA, USA

List of Contributors ix

K. G. JarvisOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Susan JosephSchool of Science and TechnologyNottingham Trent UniversityNottingham, UK

Lee S. KatzEnteric Diseases Laboratory Branch, Division of Foodborne, Waterborne and Environmental DiseasesNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and PreventionAtlanta, GA, USA

Dong Wook KimDepartment of Pharmacy, College of PharmacyHanyang UniversityKyeonggi-do, Korea

Stephen J. KnabelDepartment of Food ScienceThe Pennsylvania State UniversityUniversity Park, PA, USA

M. H. KotharyOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Sara LomonacoDepartment of Veterinary SciencesUniversità degli Studi di TorinoGrugliasco, Italy

Carolina LúquezEnteric Diseases Laboratory Branch, Division of FoodborneWaterborne and Environmental DiseasesNational Center for Emerging and Zoonotic Infectious DiseasesCenters for Disease Control and PreventionAtlanta, GA, USA

x List of Contributors

Aaron M. LynneDepartment of Biological SciencesSam Houston State UniversityHuntsville, TX, USA

M. K. MammelOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Shannon D. ManningDepartment of Microbiology and Molecular GeneticsMichigan State UniversityEast Lansing, MI, USA

Susan E. MaslankaEnteric Diseases Laboratory Branch, Division of FoodborneWaterborne and Environmental DiseasesNational Center for Emerging and Zoonotic Infectious DiseasesCenters for Disease Control and PreventionAtlanta, GA, USA

B. A. McCardellOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Ute MesselhäusserBavarian Health and Food Safety AuthorityOberschleißheim, Germany

Daniele NuceraDepartment of Agricultural, Forest and Food SciencesUniversità degli Studi di TorinoGrugliasco, Italy

Omar A. OyarzabalIEH Laboratories and Consulting Group,Seattle, WA, USA

Mário RamirezInstituto de Microbiologia, Instituto de Medicina MolecularFaculty of Medicine, University of LisbonLisboa, Portugal

List of Contributors xi

Brian H. RaphaelEnteric Diseases Laboratory Branch Division of FoodborneWaterborne and Environmental DiseasesNational Center for Emerging and Zoonotic Infectious DiseasesCenters for Disease Control and PreventionAtlanta, GA, USA

Mohana RayDepartment of ChemistryUniversity of Wisconsin-MadisonMadison, WI, USA

V. SathyamoorthyOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

David C. SchwartzLaboratory for Molecular and Computational GenomicsDepartment of ChemistryLaboratory of GeneticsUniversity of Wisconsin-MadisonMadison, WI, USA

Pallavi SinghDepartment of Microbiology and Molecular GeneticsMichigan State UniversityEast Lansing, MI, USA

M. D. SolomotisOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Insook SonCenter for Food Safety and Applied NutritionFood and Drug AdministrationCollege Park, MD, USA

David A. SweatShelby County Health DepartmentMemphis, TN, USA

xii List of Contributors

Deborah F. TalkingtonEnteric Diseases Laboratory Branch, Division of FoodborneWaterborne and Environmental DiseasesNational Center for Emerging and Zoonotic Infectious DiseasesCenters for Disease Control and PreventionAtlanta, GA, USA

Ben D. TallOffice of Applied Research and Safety AssessmentCenter for Food Safety and Applied Nutrition U.S. Food and Drug AdministrationLaurel, MD, USA

Wei ZhangInstitute for Food Safety and HealthIllinois Institute of TechnologyBedford Park, IL, USA

3

Molecular typing of foodborne pathogens has become an indispensable tool in epidemiological studies. Thanks to these techniques, we can have a better understanding of the distribution and appearance of bacterial foodborne dis-eases and have a deeper knowledge of the type of food products associated with the major foodborne pathogens. Within the molecular techniques, DNA-based techniques have prospered for more than 40 years and have been incor-porated in the first surveillance systems to monitor bacterial foodborne pathogens in the United States and other countries. However, DNA tech-niques vary widely, from techniques based on amplification of selected seg-ments of the DNA to the latest whole genome sequencing analysis. Because of the wide array of available techniques and the different results they generate, we have compiled in Section I the different DNA techniques in use for the typing of bacterial foodborne pathogens. This section covers the following techniques: (i) pulsed-field gel electrophoresis, the main typing technique at the molecular subtyping network for foodborne bacterial disease surveillance (PulseNet) by the Centers for Disease Control and Prevention (CDC); (ii) multilocus sequence typing, a very powerful technique to study bacterial population structures and changes; and (iii) high-throughput sequencing techniques that are poised to be the predominant techniques in the near future. In Section I, we have also included chapters on the analysis of results obtained with band-migration techniques, the databases and internet applications available as repository of data produced by these techniques, and the application of these molecular techniques to outbreak detection and public heath surveillance.

Section II deals with the application of techniques to type the most impor-tant bacterial foodborne pathogens. Here the emphasis is placed on the path-ogen, and each chapter describes some of the most appropriate techniques for typing each bacterial pathogen. As techniques progress and as we have better access to automated and robust techniques to study proteins, it is expected that DNA techniques will be used in association with other

Preface

xiv Preface

protein-based techniques or as first screening techniques. Until then, the techniques presented in this book are the most powerful techniques to study the molecular epidemiology of bacterial foodborne pathogens.

Omar A. OyarzabalSeattle, WA, USA

Sophia KathariouRaleigh, NC, USA

Section I

Typing Method, Analysis, and Applications

DNA Methods in Food Safety: Molecular Typing of Foodborne and Waterborne Bacterial Pathogens, First Edition. Edited by Omar A. Oyarzabal and Sophia Kathariou. © 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

1Polymerase Chain Reaction-Based Subtyping MethodsYi Chen and Insook SonCenter for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA

Polymerase chain reaction (PCR)-based molecular subtyping methods have been developed and applied to study the population genetics and molecular epidemiology of foodborne pathogens for more than two decades. These methods are based on PCR reaction and subsequent analysis of the banding pattern in gel electrophoretic. Some methods involve restriction digestion and ligation. The principles and performance (discriminatory power, epide-miological concordance, ease of use, reproducibility, typeability, etc.) of some typical PCR-based molecular subtyping methods are discussed in the following text.

Randomly amplified polymorphic DNARandomly amplified polymorphic DNA (RAPD) technique is a PCR tech-nique widely used for subtyping various bacterial pathogens. It was first described by Williams et al. (1990) and unlike conventional PCR arbitrary PCR primers are used and the target PCR products of RAPD are unknown. The primers are usually 9–10 bp long and are arbitrarily chosen by the researcher or can be randomly generated by computers. The arbitrary primer can simulta-neously anneal to multiple sites in the whole genome under low stringent conditions. When the two primers anneal within a few kilobases of each other

4 Ch 1 Polymerase Chain reaCtion-Based suBtyPing methods

in the proper direction, a fragment is amplified. These products can then be separated by gel electrophoresis and the banding patterns of different isolates compared. The genomic locations where these primers anneal are usually specific to a genotype and thus RAPD patterns are used for subtyping purposes. The annealing of arbitrary primers can be affected by only a few nucleotide differences. Because of its marked sensitivity, RAPD PCR has proven useful for differentiating both Gram-positive and Gram-negative bacteria, especially for closely related species or epidemiologically related strains (Hadrys, Balick, and Schierwater, 1992; Power, 1996; Milch, 1998) (Figure 1.1).

RAPD has been widely used to subtype various foodborne pathogens such as Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7. Nilsson et al. (1998) developed and optimized a RAPD subtyping method for Bacillus cereus that showed excellent reproducibility. Mazurier et al. (1992) investi-gated the epidemiologic relevance of RAPD using well-characterized out-break isolates of L. monocytogenes and found that RAPD correctly classified 92 out of 102 isolates into corresponding epidemic groups. Aguado, Vitas, and García-Jaloń (2001) performed RAPD and serotyping analysis to study the cross-contamination of L. monocytogenes in processed food products. Using RAPD, the authors illustrated that the strains isolated from different meat type and brand on the same date had identical subtypings, suggesting cross-contamination. The authors also found that RAPD and serotyping results were concordant, but RAPD demonstrated higher discriminatory power. The authors finally concluded that RAPD was an easy method that could be used to identify cross-contamination in post-processing environment. Vogel et al. (2001) analyzed 148 L. monocytogenes strains from vacuum-packed cold-smoked salmon produced in 10 different smokehouses using RAPD with 4 different primers separately. The authors demonstrated that RAPD provided higher discriminatory power than ribotyping and serotyping for epidemiologic typing of L. monocytogenes; however, the discriminatory power of RAPD was not as good as pulsed field gel electrophoresis (PFGE) and amplified fragment

DNA template

No amplicon Amplicon 1 Amplicon 2

Arbitrary primer

Figure 1.1 randomly amplified polymorphic dna analysis using arbitrary primers. arbitrarily designed short primers (8–12 nucleotides) anneal to a large template of genomic dna. When two primers anneal in the opposite direction to two genomic locations that are reasonably dis-tant from each other, a fragment is amplified. these randomly amplified fragments are then analyzed by gel electrophoresis, resulting in a different pattern of amplified dna fragments on the gel. to enhance priming with short primers, many primers are designed with a gC content between 10 and 70% and low annealing temperatures are used.

randomly amPlified PolymorPhiC dna 5

length polymorphism (AFLP). The authors obtained 16 reproducible RAPD profiles and the clustering of isolates using the 4 primers was identical. They identified dominant RAPD types in products from each smokehouse but also found identical RAPD types in different smokehouses, and concluded that these were persistent strains in the smokehouse environment. This study was reported in 2001. It would be interesting to reanalyze those strains with iden-tical RAPD types from different smokehouses using other discriminatory methods that were developed in the last decade. In an earlier study conducted in Japan (Yoshida et al., 1999), researchers analyzed 20 epidemiologically unrelated L. monocytogenes strains isolated from different animals and loca-tions and on different dates, and identified 18 types by RAPD using 4 primers. They also analyzed seven epidemiologically related L. monocytogenes strains isolated from raw milk and a bulk tank on a dairy farm and showed that those strains had the same RAPD type. The results demonstrated that RAPD was epidemiologically concordant. O’Donoghue et al. (1995) used RAPD to study the diversity of L. monocytogenes of different serotypes and the authors reported that serogroup 1/2 of L. monocytogenes strains are genetically more diverse than serogroup 4, a finding that was confirmed by many other subtyp-ing methods. Kim et al. (2005) studied a set of E. coli O157:H7 strains using RAPD and discovered that RAPD could not differentiate O157 strains that varied in the degree of virulence. Another study of E. coli O157:H7 (Vidovic, Germida, and Korber, 2007) demonstrated that RAPD yielded excellent dis-criminatory power for differentiating E. coli O157:H7 from animal sources.

Reproducibility is one of the biggest concerns of RAPD. Certain factors such as DNA quality and concentration, the type of Taq polymerase employed, and PCR reaction conditions can all affect the reproducibility of RAPD PCR. Therefore, it is critical to maintain the greatest consistency in DNA template quality, reagent selection, and experimental design for successful RAPD PCR. In addition, because the arbitrary primers are not specifically designed for certain genomic loci, the hybridization of the primers to the genome can be partial, which confound the PCR reaction. A RAPD protocol used by Nath, Maurya, and Gulati (2010) had only 40% reproducibility when subtyp-ing Salmonella typhi strains isolated from typhoid patients between 1987 and 2006 in India. Penner et al. (1993) conducted an inter-laboratory reproduc-ibility study of RAPD protocols with different primers and found two major variables with RAPD. One variable was that small and large polymorphic fragments were not always reproduced and therefore the size ranges of DNA fragments were different among the laboratories. The other major variable was that reproducible results were obtained with only four of the five primers using the same reaction conditions. These results highlight the importance of protocol optimization and the maintenance of consistent thermal cyclers among different laboratories when performing RAPD. Davin-Regli et al. (1995) demonstrated that variations in the concentration of template DNA

6 Ch 1 Polymerase Chain reaCtion-Based suBtyPing methods

could significantly affect the reproducibility of RAPD banding patterns. Bidet et al. (2000) evaluated three RAPD protocols using different single primers each for subtyping Clostridium difficile, and the reproducibility were only 88, 67, and 33% for the three primers. Due to the very low reproducibility of RAPD, the authors cautioned that the discriminatory power might be an overestimation.

Amplified fragment length polymorphism (AFLP)AFLP is a highly discriminatory subtyping method used for molecular sub-typing. With AFLP, genomic DNA is purified and digested with two restriction enzymes and then two different restriction-specific adaptors are ligated to ends of the restriction fragments (Figure 1.2). PCR primers, which are com-plementary to the adaptors, are designed to selectively amplify a subset of the ligated restriction fragments under stringent PCR conditions. In order to further select a subset of fragments to amplify, PCR primers are usually designed with a specific base or doublet or triplet of bases adjacent to either restriction site, and thus only the subset of genomic fragments that have matching bases adjacent to the restriction sites are amplified. PCR ampli-cons are then analyzed by gel electrophoresis, and gel patterns (polymor-phisms between and within restriction sites) are used to assign subtypes (Savelkoul et al., 1999; Foley et al., 2004; Foley, Zhao, and Walker, 2007; Singh and Mohapatra, 2008).

DNA template

Digestion with two restriction enzymes

Ligation with adaptors

Selective PCR amplification

Figure 1.2 amplified fragment length polymorphism analysis. a dna template is first digested with two restriction enzymes, preferably a hexa-cutter and a tetra-cutter; and then the restriction fragments are ligated to the adaptors. Primers are designed to be complementary to the adapter and restriction site sequences, and their 3′ ends were added by a random nucleotide for selective amplification. amplicons of selective amplification are visualized by gel electrophoresis.

amPlified fragment length PolymorPhism (aflP) 7

AFLP generally yields excellent discriminatory power, which is comparable to PFGE, the current gold standard, except for a few cases described in the following paragraph. However, AFLP is more time consuming due to the extra ligation-mediated PCR procedure. The selective PCR step could gen-erate some randomness and thus affects the reproducibility of AFLP. Internal variability due to incomplete digestion and/or ligation is also known to affect the final banding patterns.

Ripabelli, McLauchin, and Threlfall (2000) and Guerra, Bernardo, and McLauchlin (2002) developed AFLP schemes for subtyping L. monocyto-genes. They found that although not discriminatory enough, AFLP results were congruent with serotyping, phage typing, and other subtyping methods, confirming the three genetic lineages of L. monocytogenes. Keto-Timonen et al. (2007) subsequently improved AFLP by careful selection of restriction enzymes. The discriminatory power of their AFLP scheme was over 0.99 when using Simpson’s index of diversity and the results were congruent with PFGE. Lomonaco et al. (2011) compared 2 AFLP methods with PFGE for subtyping 103 unrelated L. monocytogenes strains isolated from different environmental and food sources in Italy. The authors found that the two AFLP methods and PFGE had similar discriminatory power. However, one AFLP method suffered from unsatisfactory typeability for certain strains from dairy products. This AFLP method uses restriction enzyme Sau3AI; therefore, it has been suggested that some L. monocytogenes strains from dairy products are not restricted with Sau3AI, which is possibly due to the methylation of cytosine at GATC. Therefore, careful selection of restriction enzyme is very critical for the typeability of AFLP. Herrera et al. (2002) reported a discrepancy in the relatedness of the Shigella flexneri strain typed by plasmid profiling, serotyping, and AFLP analysis, and no definitive con-clusions were drawn about the epidemiologic concordance of AFLP with this work.

Since the invention of the original AFLP scheme, modifications of this technique have been developed. One example is the fluorescent amplified fragment length polymorphism (FAFLP). With this technique, the ampli-fied restricted fragments are labeled with fluorescent molecules and there-fore the detection of banding patterns is of higher resolution than the traditional gel electrophoretic patterns. Ross and Heuzenroeder (2005) compared FAFLP and PFGE using a set of Salmonella enterica serovar Typhimurium DT126 isolates from several foodborne outbreaks in Australia. The authors found that AFLP had slightly higher discriminatory power than PFGE. While both methods successfully clustered isolates within an outbreak, some unrelated isolates could not be differentiated from outbreak isolates by either method. In addition to FAFLP, Lan and Reeves (2007) also reported a radioactively labeled AFLP method for subtyping S. enterica.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Herrera%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

8 Ch 1 Polymerase Chain reaCtion-Based suBtyPing methods

Repetitive-sequence-based PCRRepetitive-sequence-based PCR (rep-PCR) typing is another subtyping technique that utilizes PCR. In bacteria, there are dispersed chromosomal repetitive elements that are randomly distributed throughout the genomes (Versalovic, Koeuth, and Lupski, 1991a) (Figure 1.3). These sequences dif-fer in size and do not encode proteins. With rep-PCR, primers that are complementary to these repeats are designed and used to amplify differ-ently sized DNA fragments lying between the repeats. One type of repeat sequence is repetitive extragenic palindromes (REPs), which are regulatory sequences within untranslated regions of bacterial operons. REPs were ini-tially discovered in Salmonella and E. coli (Gilson et al., 1984; Gilson et al., 1990; Sharples and Lloyd, 1990; Ridley, 1998). The family of REPs com-prises short DNA segments, generally 30–40 bp, include inverted repeats, and there are around 500–1000 copies per genome (Stern et al., 1984). They can appear as a single copy or as multiple adjacent copies and occupy up to 1% of the genomes of Salmonella and E. coli. Jersek et al. (1996) employed previously developed primers and found that Listeria spp. also possess short REP elements.

Another type of repetitive sequences is named as enterobacterial repeti-tive intergenic consensus (ERIC) sequence. ERIC sequences are widely dis-tributed across a wide range of species and were originally described in Salmonella, E. coli, and other members of Enterobacteriaceae. The ERIC sequence is a palindrome of 127 bp that contains a conserved central inverted repeat. Variations of ERIC sequences include shorter sequences caused by internal deletions and longer sequences caused by insertions. They are mostly present in intergenic regions of the genome. The number of copies of ERIC sequences ranges from around 30 copies in E. coli to around 150 copies in Salmonella Typhimurium and over 700 copies in some other Enterobacteria-ceae (Hulton, Higgins, and Sharp, 1991; Burr, Josephson, and Pepper, 1998). ERIC PCR was first described by Versalovic, Koeuth, and Lupski (1991b). Jersek et al. (1996) subsequently found ERIC sequences in Listeria spp. and found that rep-PCR showed a higher discriminative power than ERIC-PCR for subtyping closely related strains of L. monocytogenes.

Primer

GenomeRepeatsequence

PCR

Figure 1.3 repetitive-sequence-based PCr. Primers are designed to bind to the repetitive elements and regions between these repeats are amplified. these fragments are then analyzed by gel electrophoresis.

rePetitive-sequenCe-Based PCr 9

Another class of repeats, BOX, was originally found in Streptococcus pneumonia (van Belkum and Hermans, 2001). BOX sequences are also intergenic regions that form stem-loop structures. They are mosaic repetitive elements that include combinations of three subunits, BOX-A (59 bp), BOX-B (45 bp), and BOX-C (50 bp). The evolutionary origin and functions of these BOX regions remain unclear, and they are not related to REP and ERIC sequences. These regions have proven useful for the differentiation of enteric species and the development of strain-specific subtyping methods (Weigel et al., 2004; Cesaris et al., 2007). Both REP and ERIC sequences con-tain conserved regions for primer targeting and variable regions for poly-morphism detection. For example, REP primers usually target the left and right sides of a conserved palindromic sequence and are oriented in opposite directions so that the primer extends outwardly in a 3′ direction away from the palindrome. The regions between the repetitive palindromic islands were thus amplified. These regions range in size from 200 bp to 4 kbp and provide a unique chromosomal pattern for a given strain. In a rep-PCR design, mul-tiple primers or one single oligoprimer can be used. A list of these primers is provided in Table 1.1.

Van Kessel et al. (2005) analyzed 61 L. monocytogenes strains from raw milk using an automated rep-PCR system. The results showed that rep-PCR clusters correlated with species and serotypes of Listeria spp. Jersek et al. (1999) developed a rep-PCR scheme targeting short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic con-sensus (ERIC) sequences in L. monocytogenes and found that these tech-niques have a high discriminatory power (0.98). One advantage of rep-PCR is that it can be automated due to the simple PCR operation. However, rep-PCR suffered from low discriminatory power when subtyping L. monocyto-genes strains from the same serotype, and it could not discriminate between serotypes 1/2b and 4b strains. Zunabovic et al. (2012) evaluated the potential

Table 1.1 Primers and PCr conditions for common repetitive elements

oligo name/sequenceannealing temperature

extension temperature references

reP1r-i/iiiiCgiCgiCatCiggC 40 65 versalovic, Koeuth, and lupski (1991a)reP2-i/iCgiCttatCiggCCtaC

eriC1r/atgtaagCtCCtggggattCaC 52 65 versalovic, Koeuth, and lupski (1991a)eriC2/aagtaagtgaCtggggtgagCg

BoXa1r/CtaCggCaagCggaCgCtgaCg 52 65 Proudy et al. (2008)mBo1/CCgCCgttgCCgCCgttgCCgCCg 54 65 versalovic, Koeuth, and

lupski (1991a)gtgs/gtggtggtggtggtg 40 65 versalovic, Koeuth, and

lupski (1991a)oPa-1/CaggCCCttC 35 72 enger et al. (2001)

Click to search for citations by this author.

10 Ch 1 Polymerase Chain reaCtion-Based suBtyPing methods

of three rep-PCR methods (GTG₅ and REPI+II) for the typing of Listeria spp. including L. monocytogenes from a cold-smoked salmon production facility and compared rep-PCR methods with PFGE. The authors found that although rep-PCR yielded a lower discriminatory power than PFGE, it was still a useful tool for tracing contamination niches and transmission routes of Listeria spp. in the food processing environment. However, it is important to note that this study evaluated rep-PCR using a set of Listeria spp. that included six species. A close examination of their data showed that the discriminatory power of rep-PCR within L. monocytogenes was still limited. Hahm et al. (2003) suggested that rep-PCR and BOX-PCR can serve as first step screening prior to PFGE for subtyping E. coli O157. The authors also found differences between the strain relatedness identified by each method, but the subtype profiles of the E. coli O157:H7 isolates were virtually identical using rep-PCR and BOX-PCR. Nath, Maurya, and Gulati (2010) analyzed a collection of S. enterica serotype Typhi strains isolated from typhoid patients between 1987 and 2006 using ERIC PCR and concluded that ERIC-PCR was very efficient with excellent discriminatory power and reproducibility.

A modification of rep-PCR is the incorporation of fluorescently labeled primers where amplified products are visualized by a fluorescence-based DNA analyzer. Del Vecchio et al. (1995) described a fluorescence-enhanced rep-PCR for subtyping Staphylococcus aureus. This modification reduces the labor required for manual gel electrophoresis and simplifies visualization, comparison, and storage of DNA banding patterns. Because rep-PCR is very simple, and does not require extra restriction or ligation steps like other PCR-based subtyping methods, rep-PCR can be easily automated. Brusetti et al. (2008) developed a florescent-BOX-PCR for subtyping E. coli and B. cereus and the authors concluded that the increased resolution power by using florescence-labeled oligos detected up to 12 times more fragments than traditional BOX-PCR, and thus improved the discriminatory power. Healy et al. (2005) described a modification using a commercially available automated rep-PCR system. The automated system significantly improves the reproduc-ibility of rep-PCR over the manual operations. The built-in software pro-grams improved image analyses, but images must be captured and imported prior to analysis and subjectivity remains because optimization parameters can be modified. The automated system has obvious advantages over PFGE and MLST, both of which require skilled operators.

Despite the reports of the success of ERIC-PCR for subtyping various bac-terial species, some scientists cast doubt on the performance of ERIC-PCR. One disadvantage of ERIC-PCR is that the distributions of some ERIC sequences limit the potential of ERIC-PCR as a subtyping tool. In order to understand the performance and mechanism of ERIC-PCR, Wilson and Sharp (2006) studied the distribution and evolution of ERIC sequences in

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Hahm%20BK%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus

multiPle-loCus variaBle-numBer tandem rePeat analysis 11

several Enterobacteriaceae species. The authors found that the copy numbers and locations of ERIC sequences vary greatly among different strains within the same species, which serve as the base of discriminatory ability of ERIC-PCR. However, the authors observed that some E. coli strains do not have sufficient full length ERIC sequences and the number of amplified fragments is not sufficient to generate meaningful patterns for subtyping purposes. Another disadvantage of ERIC-PCR is that the reaction appeared to be not specific. In a detailed investigation, Gillings and Holley cautioned that ERIC-PCR would generate amplified products even from genomes that do not pos-sess any ERIC sequences (Gillings and Holley, 1997). Other scientists reported similar findings. For example, Niemann et al. (1999) used ERIC-PCR to subtype strains of Sinorhizobium meliloti, a member of Proteobacteria that usually do not have ERIC sequences. The authors sequenced part of the amplified fragments and found that they did not match ERIC sequences; however, the resulting banding patterns were still able to serve subtyping pur-poses. Wei et al. (2004) also reported that the amplified fragments by their ERIC-PCR protocol showed no similarity to ERIC sequences. This indicated that the short primers might bind to nonspecific regions. Therefore, it appeared that the ERIC primers work as arbitrary primers just like those primers used in RAPD. Wilson and Sharp (2006) stated that we should revisit earlier con-clusions that many bacteria species contain ERIC sequences based on positive ERIC-PCR amplifications. Deplano et al. (2000) described a multicenter evaluation of epidemiological typing of methicillin-resistant S. aureus strains by repetitive-element PCR analysis, a study conducted by the European Study Group on Epidemiological Markers of the European Society of Clinical Microbiology and Infectious Diseases. The study used PFGE as the reference method and showed that rep-PCR had lower discriminatory power insuffi-cient interlaboratory reproducibility. The authors concluded that it was diffi-cult to fully standardize rep-PCR assays.

Multiple-locus variable-number tandem repeat analysisMultiple-locus variable-number tandem repeat analysis (MLVA) is one of CDC’s candidates for complimenting PFGE for epidemiological subtyping. MLVA targets tandem repeat (TR) polymorphisms in the genomes of differ-ent bacterial pathogens (Hyytia-Trees et al., 2010). PCR primers are designed to amplify all possible tandem repeats (TRs) in the chromosome based on whole genome sequences. The size and number of repeats at each loci are then analyzed by computer and combinations of these repeats define MLVA types (Table 1.2). Tandem repeats are well recognized as containing phyloge-netic signals. The repeats sometimes are targets of evolutionary events such

12 Ch 1 Polymerase Chain reaCtion-Based suBtyPing methods

as mutation and recombination and these evolutionary events may change the size and number of the repeats. The number of such repeats at a specific locus is similar among isolates that are closely related and varies between unrelated isolates. TRs correlate with many genomic changes essential for bacterial survival under stress conditions. Such changes include deletions; insertions and mutations that affect gene regulation; antigenic shifts; and inactivation of mismatch repair systems (Ramazanzadeh and McNerney, 2007). TRs actually play an important role in the adaptation of bacteria, espe-cially those with small genomes. Therefore, MLVA is expected to provide relatively accurate information about the genetic relatedness of different bac-terial strains. Unlike PFGE, the targets of MLVA are specific TRs that can be PCR amplified using primers designed based on whole genome sequences. Thus, MLVA is easier to interpret than PFGE, because the fragments gener-ated by MLVA are of known size and sequence. In addition, the essential steps in MLVA are multiplex PCR and capillary electrophoresis, which are very easy to perform, standardize, and automate, making MLVA a potentially high-throughput subtyping method (Lindstedt, 2005; Lindstedt et al., 2008). The final results in MLVA are sizes of each TR loci and thus it is easier to compare than gel-banding patterns generated by other fragment-based methods. The key to the development of reliable and accurate MLVA schemes is the identification of TRs. For instance, one of the limitations associated

Table 1.2 select vntr loci in L. monocytogenes identified in the literature

vntr locus Copy no. repeat locus tag references

lm-2 11–20 ttgtat lmo0582 sperry et al. (2008)

lm-3 1–9 taaaaCCta lmo0842 sperry et al. (2008)lm8 3–4 CagCtttCtCagCag lmo1941 sperry et al. (2008)lm10 3–9 gaagaaCCaaaa lmo0220 sperry et al. (2008)lm11 1–6 ttgCttgttttg lmo0320 sperry et al. (2008)lm15 1–7 CaaaagataCaC lmo0627 sperry et al. (2008)lm23 15–42 CatCgg lmo1799 sperry et al. (2008)lm-32 13–21 aaCaCC lmo1290 sperry et al. (2008)tr-1 17 CCggtagat lmo1136 miya et al. (2008)tr-2 11 CatCgg lmo1799 miya et al. (2008)tr-3 4 tCa lmo0186 miya et al. (2008)lm-tr 1 4–5 taaaaCCta lmo0842 murphy et al. (2007)lm-tr 2 2–3 tatttttatttaaaaatg lmof2365_2121 murphy et al. (2007)

lmof2365_2122lm-tr 3 13–14 CCggtagat lmof2365_1144 murphy et al. (2007)

lmof2365_1145lm-tr 4 2–3 gaagaaCCaaaa lmof2365_0231 murphy et al. (2007)

lmof2365_0232lm-tr 5 20–21 gtagatCCg lmo1136 murphy et al. (2007)lm-tr 6 3 CCagaCCCaaCa lmo1289 murphy et al. (2007)

multiPle-loCus variaBle-numBer tandem rePeat analysis 13

with the development of MLVA for Salmonella is that different serovars dif-fer slightly in their genomic organizations and thus some well-characterized TRs may not be present in all serovars. In this case, serovar-specific MLVA typing schemes have been developed (Ross and Heuzenroeder, 2005; Ross et al., 2011). Large amounts of complete genome sequence data are essential for developing MLVA schemes and PFGE is superior to MLVA in this aspect since no prior knowledge of the whole genome sequence data is required with PFGE (Karama and Gyles, 2010; Kruy, van Cuyck, and Koeck, 2011; Sobral et al., 2012). A list of TRs used to develop MLVA strategies for L. monocytogenes are listed in Table 1.3.

Another important feature for the development of reproducible and epi-demiologically relevant MLVA schemes is the stability of the targets. Some TRs can be very unstable and potentially separate isolates within the same outbreak clone, which would confound the study of long-term epidemiology. Some extremely unstable TRs may even change during regular laboratory culturing and affect the reproducibility of MLVA. Another potential draw-back of MLVA is that the primers for amplifying the TRs are designed based on the relatively small number of whole genome sequences currently avail-able. Consequently, typeability may become a limiting factor because not all TRs from strains of the same species may be successfully amplified. For example, an insertion within a TR would confound the analysis of the size of the TR. Therefore, the selection of TRs for MLVA typing and design of PCR primers are critical to an epidemiologically relevant MLVA scheme. Intensive evaluation and validation are needed for each MLVA scheme.

MLVA has been applied to many foodborne pathogens such as E. coli, Salmonella spp., and L. monocytogenes and has been proven to yield very high discriminatory power. Hyytia-Trees et al. (2006) evaluated the epidemi-ologic relevance of a MLVA scheme for E. coli O157:H7 and claimed MLVA had promising epidemiologic relevance by correctly clustering isolates belonging to eight well-characterized outbreaks. In 2006, a MLVA scheme for subtyping L. monocytogenes was described by Murphy et al. (2007). In that study, MLVA was shown to discriminate isolates of the same serotype and correlate with PFGE data from the same set of isolates. Kawamori et al. (2008) compared MLVA and PFGE for subtyping E. coli O157:H7 and found that there was a good correlation between MLVA and PFGE.

Although MLVA is a fragment-based method, the utilization of meaningful molecular markers, PCR, and capillary electrophoresis generate a more phy-logenetically meaningful and nonambiguous output, which provide a major evolution over other fragment-based subtyping methods. Ross et al. (2009) evaluated MLVA and PFGE for subtyping Salmonella Enteritidis, and the discriminatory indexes were 0.968 and 0.873, respectively. These studies dem-onstrated the great potential of MLVA for reliable and rapid subtyping of foodborne pathogens.

14 Ch 1 Polymerase Chain reaCtion-Based suBtyPing methods

Tabl

e 1.

3 Co

mpa

riso

n of

maj

or s

trai

n ty

ping

met

hods

in t

erm

s of

per

form

ance

on

vario

us c

rite

ria

subt

ypin

g m

etho

dsta

rget

sty

pe o

f da

ta o

utpu

tdi

scri

min

ator

y po

wer

epid

emio

logi

c co

ncor

danc

ere

prod

ucib

ility

type

abili

tyCo

st

RAPD

Poly

mor

phis

m w

ithin

and

bet

wee

n ar

bitr

ary

prim

ing

regi

ons

dna

gel-

band

ing

patt

ern

mod

erat

em

ediu

mm

ediu

mh

igh

med

ium

rep-

PCR

repe

titi

ve e

lem

ents

dna

gel-

band

ing

patt

ern

mod

erat

em

ediu

mm

ediu

mh

igh

low

MLV

Avn

trnu

mbe

rh

igh

hig

hm

ediu

m t

o hi

ghh

igh

med

ium

AFLP

Poly

mor

phis

m w

ithi

n /b

etw

een

rest

ricti

on s

ites

dna

gel-

band

ing

patt

ern

mod

erat

e to

hig

hm

ediu

mm

ediu

m t

o hi

ghh

igh

med

ium

PCR-

RFLP

Poly

mor

phis

m w

ithi

n/be

twee

n re

stric

tion

sit

es in

spe

cific

gen

esdn

a ge

l-ba

ndin

g pa

tter

nm

oder

ate

med

ium

hig

hh

igh

med

ium