Pulse Field Gel Electrophoresis: Methods and Protocols [1 ed.] 1493925989, 9781493925988

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Methods in Molecular Biology 1301

Kieran Jordan Marion Dalmasso Editors

Pulse Field Gel Electrophoresis Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series editor John M.Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Pulse Field Gel Electrophoresis Methods and Protocols Edited by

Kieran Jordan Teagasc Food Research Center, Moorepark, Fermoy, Co. Cork, Ireland

Marion Dalmasso Department of Microbiology & Alimentary Pharmabiotic Centre, University College Cork, Co. Cork, Ireland

Editors Kieran Jordan Teagasc Food Research Center, Moorepark Fermoy, Co. Cork, Ireland

Marion Dalmasso Department of Microbiology & Alimentary Pharmabiotic Centre University College Cork Co. Cork, Ireland

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2598-8 ISBN 978-1-4939-2599-5 (eBook) DOI 10.1007/978-1-4939-2599-5 Library of Congress Control Number: 2015935078 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com)

Preface First described in 1984 for the separation of large DNA fragments, pulsed field gel electrophoresis (PFGE) has emerged as the “gold standard” method for molecular subtyping of bacteria. Comparative analysis of different isolates of the same bacterial species is important for pathogenic and nonpathogenic bacteria. For pathogenic bacteria, PFGE has revolutionized epidemiological investigations for disease surveillance studies and contributed significantly to disease monitoring and control programs, facilitating the tracking of infection and contamination. For nonpathogenic bacteria, PFGE can be used for monitoring survival of particular strains or tracking contamination routes, among other things. The success of PFGE is based on its discriminatory power, reproducibility across different laboratories (with harmonized protocols), and relatively low cost. The use of whole-genome sequencing for routine disease monitoring is gaining in importance, yet despite this, PFGE will continue as an important tool in many laboratories, and from a disease surveillance perspective, it will remain as a tool in association with genomics and metagenomics. Harmonized methodologies, for analysis and interpretation of profiles are key to the success of PFGE. This has been demonstrated with the success of the PulseNet protocols, facilitating international comparisons of PFGE profiles of disease-causing bacteria. This book, which will be of interest to epidemiologists, food microbiologists, and anyone working on comparing bacterial isolates, whether pathogenic or nonpathogenic, will advance the harmonization of PFGE methodologies and facilitate interlaboratory comparisons of PFGE profiles from pathogenic and nonpathogenic bacteria. Fermoy, Co. Cork, Ireland Co. Cork, Ireland

Kieran Jordan Marion Dalmasso

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Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

INTRODUCTION TO PFGE

1 Pulsed-Field Gel Electrophoresis for Disease Monitoring and Control . . . . . . . John Besser 2 Harmonization of PFGE Profile Analysis by Using Bioinformatics Tools: Example of the Listeria monocytogenes European Union Reference Laboratory Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benjamin Félix, Sophie Roussel, and Bruno Pot 3 PFGE as a Tool to Track Listeria monocytogenes in Food Processing Facilities: Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marion Dalmasso and Kieran Jordan 4 Case Study of the Use of Pulsed Field Gel Electrophoresis in the Detection of a Food-Borne Outbreak . . . . . . . . . . . . . . . . . . . . . . . . . . Niall De Lappe and Martin Cormican

PART II

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PFGE AND PATHOGENIC BACTERIA

5 Pulsed-Field Gel Electrophoresis for Listeria monocytogenes . . . . . . . . . . . . . . . Laura Luque-Sastre, Séamus Fanning, and Edward M. Fox 6 Pulsed-Field Gel Electrophoresis (PFGE) for Pathogenic Cronobacter Species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Qiongqiong Yan and Séamus Fanning 7 Pulsed-Field Gel Electrophoresis of Bacillus cereus Group Strains. . . . . . . . . . . Paul Drean and Edward M. Fox 8 Pulsed-Field Gel Electrophoresis of Staphylococcus aureus . . . . . . . . . . . . . . . . George R. Golding, Jennifer Campbell, Dave Spreitzer, and Linda Chui 9 The Use of Pulsed-Field Gel Electrophoresis for Genotyping of Clostridium difficile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wondwossen A. Gebreyes and Pamela R.F. Adkins 10 Molecular Subtyping of Clostridium botulinum by Pulsed-Field Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carolina Lúquez, Lavin A. Joseph, and Susan E. Maslanka 11 Pulsed-Field Gel Electrophoresis of Yersinia pestis . . . . . . . . . . . . . . . . . . . . . . Tamara Revazishvili and Judith A. Johnson 12 Pulsed Field Gel Electrophoresis of Group A Streptococci . . . . . . . . . . . . . . . . Luca Agostino Vitali, Giovanni Gherardi, and Dezemona Petrelli

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13 Application of Pulsed Field Gel Electrophoresis to Type Campylobacter jejuni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ping Zhou and Omar A. Oyarzabal 14 Pulsed-Field Gel Electrophoresis of Pseudomonas aeruginosa . . . . . . . . . . . . . . Honghua Hu and Jim Manos 15 PFGE for Shiga Toxin-Producing Escherichia coli O157:H7 (STEC O157) and Non-O157 STEC . . . . . . . . . . . . . . . . . . . . . . . Patricia Jaros, Muriel Dufour, Brent Gilpin, Molly M. Freeman, and Efrain M. Ribot 16 Pulsed-Field Gel Electrophoresis of Salmonella enterica . . . . . . . . . . . . . . . . . . Antonio Camarda, Elena Circella, Antonia Pupillo, Marilisa Legretto, Michele Marino, and Nicola Pugliese

PART III

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PFGE AND NON-PATHOGENIC BACTERIA

17 PFGE Protocols to Distinguish Subspecies of Lactococcus lactis . . . . . . . . . . . . Pascal Le Bourgeois, Delphine Passerini, Michèle Coddeville, Maéva Guellerin, Marie-Line Daveran-Mingot, and Paul Ritzenthaler 18 The Use of PFGE Method in Genotyping of Selected Bacteria Species of the Lactobacillus Genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tomasz Gosiewski and Monika Brzychczy-Wloch 19 Pulsed-Field Gel Electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Victoria Chuat and Marion Dalmasso 20 Pulsed Field Gel Electrophoresis for Bifidobacterium . . . . . . . . . . . . . . . . . . . . Esther Jiménez, Marta Gómez, and Laura Moles 21 Pulsed Field Gel Electrophoresis for Dairy Propionibacteria . . . . . . . . . . . . . . Victoria Chuat, Rosangela de Freitas, and Marion Dalmasso

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Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors PAMELA R.F. ADKINS • Veterinary Medicine and Surgery, University of Missouri, Columbia, MO, USA JOHN BESSER • Enteric Diseases Laboratory Branch, NCEZID/DFWED, MS C03, Centers for Disease Control and Prevention, Atlanta, GA, USA PASCAL LE BOURGEOIS • LMGM, UPS, Université de Toulouse, Toulouse, France MONIKA BRZYCHCZY-WLOCH • Jagiellonian University Medical College, Krakow, Poland ANTONIO CAMARDA • Department of Veterinary Medicine, University of Bari, Valenzano, BA, Italy JENNIFER CAMPBELL • National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada VICTORIA CHUAT • UMR1253 Science et Technologie du Lait et de l’Oeuf, CIRM-BIA, INRA, Rennes, France; UMR1253 Science et Technologie du Lait et de l’Oeuf, AGROCAMPUS OUEST, Rennes, France LINDA CHUI • Provincial Laboratory for Public Health, Edmonton, AB, Canada; Department of Laboratory Medicine and Pathology University of Alberta, Edmonton, AB, Canada ELENA CIRCELLA • Department of Veterinary Medicine, University of Bari, Valenzano, BA, Italy MICHÈLE CODDEVILLE • LMGM, UPS, Université de Toulouse, Toulouse, France MARTIN CORMICAN • School of Medicine, National University of Ireland Galway, Galway, Ireland MARION DALMASSO • Department of Microbiology & Alimentary Pharmabiotic Centre, University College Cork, Co.Cork, Ireland MARIE-LINE DAVERAN-MINGOT • LMGM, UPS, Université de Toulouse, Toulouse, France PAUL DREAN • CSIRO Food and Nutrition, Werribee, VIC, Australia MURIEL DUFOUR • Enteric Reference Laboratory, Institute of Environmental Science & Research Ltd, Upper Hutt, New Zealand SÉAMUS FANNING • UCD-Centre for Food Safety, WHO Collaborating Centre for Research, Reference & Training on Cronobacter, School of Public Health, Physiotherapy & Population Science, University College Dublin, Belfield, Dublin 4, Ireland BENJAMIN FÉLIX • ANSES, Maisons-Alfort Laboratory for Food Safety, European Union Reference Laboratory for Listeria monocytogenes, University Paris-East, Maisons-Alfort, France EDWARD M. FOX • CSIRO Food and Nutrition, Werribee, VIC, Australia MOLLY M. FREEMAN • Enteric Diseases Laboratory Branch, Centres for Disease Control and Prevention, Atlanta, GA, USA ROSANGELA DE FREITAS • Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa, MG, Brazil WONDWOSSEN A. GEBREYES • Veterinary Preventive Medicine, The Ohio State University, Columbus, OH, USA

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GIOVANNI GHERARDI • Centro Integrato di Ricerca (CIR), University “Campus Bio-Medico”, Rome, Italy BRENT GILPIN • Water Group, Institute of Environmental Science & Research Ltd, Christchurch, New Zealand GEORGE R. GOLDING • Antimicrobial Resistance and Nosocomial Infections National Microbiology Laboratory, Winnipeg, MB, Canada MARTA GÓMEZ • Departamento de Nutrición, Bromatología y Tecnología de los Alimentos. Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain TOMASZ GOSIEWSKI • Jagiellonian University Medical College, Krakow, Poland MAÉVA GUELLERIN • LMGM, UPS, Université de Toulouse, Toulouse, France LMGM, CNRS, UMR5100, Toulouse, France HONGHUA HU • Australian School of Advanced Medicine, Macquarie University, Sydney, NSW, Australia PATRICIA JAROS • Molecular Epidemiology and Public Health Laboratory, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand ESTHER JIMÉNEZ • Departamento de Nutrición, Bromatología y Tecnología de los Alimentos. Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain JUDITH A. JOHNSON • Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA KIERAN JORDAN • Teagasc Food Research Center, Moorepark, Fermoy, Co. Cork, Ireland LAVIN A. JOSEPH • Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA NIALL DE LAPPE • National Salmonella, Shigella & Listeria Reference Laboratory, Medical Microbiology Department, University Hospital Galway, Galway, Ireland MARILISA LEGRETTO • Department of Veterinary Medicine, University of Bari, Valenzano, BA, Italy LAURA LUQUE-SASTRE • UCD-Centre for Food Safety, WHO Collaborating Centre for Research, Reference & Training on Cronobacter, School of Public Health, Physiotherapy & Population Science, University College Dublin, Belfield, Ireland CAROLINA LÚQUEZ • Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA JIM MANOS • Department of Infectious Diseases and Immunology, University of Sydney, Sydney, NSW, Australia MICHELE MARINO • Department of Veterinary Medicine, University of Bari, Valenzano, BA, Italy SUSAN E. MASLANKA • Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA LAURA MOLES • Departamento de Nutrición, Bromatología y Tecnología de los Alimentos. Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain OMAR A. OYARZABAL • IEH Laboratories and Consulting Group, Lake Forest Park, WA, USA; University of Vermont Extension, Berlin, VT, USA DELPHINE PASSERINI • LMGM, UPS, Université de Toulouse, Toulouse, France DEZEMONA PETRELLI • School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, MC, Italy BRUNO POT • Applied Maths NV, Sint-Martens-Latem, Belgium NICOLA PUGLIESE • Department of Veterinary Medicine, University of Bari, Valenzano, BA, Italy

Contributors

ANTONIA PUPILLO • Department of Veterinary Medicine, University of Bari, Valenzano, BA, Italy TAMARA REVAZISHVILI • Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA EFRAIN M. RIBOT • Enteric Diseases Laboratory Branch, Centres for Disease Control and Prevention, Atlanta, GA, USA PAUL RITZENTHALER • LMGM, UPS, Université de Toulouse, Toulouse, France SOPHIE ROUSSEL • ANSES, Maisons-Alfort Laboratory for Food Safety, European Union Reference Laboratory for Listeria monocytogenes, University Paris-East, Maisons-Alfort, France DAVE SPREITZER • National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada LUCA AGOSTINO VITALI • School of Pharmacy, University of Camerino, Camerino, MC, Italy QIONGQIONG YAN • UCD-Centre for Food Safety, WHO Collaborating Centre for Research, Reference & Training on Cronobacter, School of Public Health, Physiotherapy & Population Science, University College Dublin, Belfield, Dublin 4, Ireland PING ZHOU • Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, USA

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Part I Introduction to PFGE

Chapter 1 Pulsed-Field Gel Electrophoresis for Disease Monitoring and Control John Besser Abstract Pulsed-field gel electrophoresis (PFGE) is the “gold standard” for molecular subtyping and has significant applications in disease monitoring and control programs. This chapter discusses the advantages of PFGE in light of developing technologies such as whole-genome sequencing. Keywords PFGE, Subtyping, PulseNet, Surveillance

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Introduction For over 15 years, pulsed-field gel electrophoresis (PFGE) has been the gold standard for bacterial molecular subtyping against which other methods are compared. It has revolutionized foodborne bacterial disease surveillance and has had significant applications in other disease monitoring and control programs such as those for hospital-acquired, invasive, and respiratory infections. It also has applications in other non-disease related situations, such as tracking contamination or monitoring survival of particular strains. The success of PFGE can be attributed to its versatility, reproducibility, relatively low cost, discriminatory power, and high epidemiological concordance for many pathogens of public health importance. Even as the public health community moves towards whole-genome sequencing (WGS) for routine disease monitoring functions, PFGE remains an important tool. Lessons learned over the last two decades will inform efforts into the genomic and metagenomic eras. In this chapter, the role that PFGE has played for disease monitoring and control will be described along with key benefits and limitations for this activity.

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_1, © Springer Science+Business Media New York 2015

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PFGE in public health PFGE was first described in 1984 to separate large fragments of DNA, such as those obtained by cleavage with infrequent-cutting endonucleases. It was first used to map genomes of the yeast Saccharomyces cerevisiae and the parasitic protozoa Trypanosoma brucei [1, 2]. Early efforts focused on other yeasts and parasites, the human genome, and eventually bacterial genomes. By the early 1990s, the method began migrating from research laboratories into public health laboratory and hospital infection control practice [3–7], where it was used primarily for comparison of strains obtained during outbreak investigations. In this capacity, it proved valuable in assessing the likelihood of connection between disease events that would require public health attention, such as community outbreaks or nosocomial infections. However, it was not until the mid1990s that PFGE was used for primary detection of widely dispersed clusters of disease using sporadic case surveillance. The use of subspecies identification for cluster detection and trend analysis was not new. Serotyping had been used as part of national case-based surveillance for pathogens such as Salmonella enteritica and Neisseria meningitidis for decades, allowing identification (or ruling out) of outbreaks and trends that may not have been otherwise recognized. One of the largest outbreaks was Salmonella serotype Enteritidis outbreak associated with ice cream that affected an estimated 224,000 persons throughout the lower 48 US states in 1994 [8]. Serotype information allowed researchers to focus on those cases most likely to share a common source. Restricting the case definition in this manner improves the signal to noise ratio, making it possible to detect trends that may be obscured by the large number of salmonellosis cases that normally occur. Similarly, outbreaks and population trends due to Neisseria meningitidis have been detected through serotype-specific sporadic case surveillance since at least 1977 [9, 10]. Other subspecies phenotypic markers such as antibiotic susceptibility or virulence determinants had been routinely used for such pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) or enterotoxigenic E. coli (ETEC). Could higherresolution molecular methods make it possible to identify or solve more outbreaks or detect trends with a new level of specificity, including infections for which phenotypic markers were not available or practical? PFGE was first used for real-time sporadic case surveillance in 1994 when it was demonstrated that four out of ten outbreaks of E. coli O157:H7 (O157) identified during a 2-year period in Minnesota were only detected on the basis of molecular subtype clustering [11]. The authors described similar findings with PFGEbased sporadic case surveillance for Salmonella Typhimurium, where four out of six larger community outbreaks were identified during a 4-year period that would not have otherwise been detected [12].

Pulsed-Field Gel Electrophoresis for Disease Monitoring and Control

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In 1996, the US Centers for Disease Control and Prevention (CDC) developed PulseNet, the PFGE-based US national molecular subtyping network for foodborne bacterial disease surveillance. Strict standardization and a centralized database made it possible for laboratories in the system to compare patterns and identify trends [13]. PulseNet’s official kick off was in 1998 with four participating US states. Today PulseNet operates in 82 countries around the world. Standard protocols for methicillin-resistant MRSA that would allow inter-laboratory comparisons were developed in the USA and Europe [14, 15]. The technology is used in many locations as part of investigation and control of other diseases, such as invasive Streptococcus pyogenes disease [16] and meningococcal meningitis [17]. The role that strain typing methods including PFGE play in detecting or clarifying potential epidemiological associations in diseases caused by free-living bacterial pathogens is less straightforward than the role of similar “DNA fingerprinting” methods for human forensics. Many pathogens have an independent existence from their human host for at least part of their life cycle, and complex and possibly unknowable routes of transmission may be involved. Furthermore, events leading to foodborne, waterborne, or environment-associated outbreaks form a spectrum in terms of clonality, ranging from true point source events involving a single clone to massive fecal contamination involving many microbial strains, species, genera, or even kingdoms. This can make interpretation of individual results difficult in some circumstances. Nevertheless, when used appropriately it can have great public health benefit. The most basic interpretation of PFGE pattern groupings is that cases with indistinguishable strain patterns are more likely to share an epidemiological association than cases with differing PFGE patterns. When cases are grouped together in this manner, the likelihood of identifying a common source leading to public health action is greatly increased. Unlike DNA typing for human forensics, microbial subtyping is primarily used as a population-based analysis tool. PFGE data constitutes only part of the overall epidemiological evidence used for establishing associations between exposures and illness. As a standard method for disease monitoring and control, PFGE has strengths and limitations. While most subspecies classification methods require extensive agent-specific development, the same basic PFGE protocol and instrumentation can be used for most culturable organisms. The method is readily standardized, reproducible, robust, and has generally exhibited good epidemiological concordance. Laboratory methods vary greatly in terms of their ability to resolve differences within bacterial species, ranging from no resolution (only species identification performed, all cases classified together) to whole-genome sequencing with long-read technology and epigenetic markers (all cases are different). For many

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pathogens, PFGE represents a simple compromise between these extremes. As with any all-purpose tool this works better for some functions than others. For example, a single unresolvable PFGE subtype (JEGX01.0004) compromises 46 % of the PulseNet USA Salmonella Enteritidis database, the remainder being comprised of 886 subtypes (personal communication, Beth Tolar of US Centers for Disease Control and Prevention). This subtype has been responsible for a wide range of outbreaks from varying sources, but detection and investigation of clusters is somewhat compromised by the inability to separate unrelated cases that normally occur in the background from true outbreak cases. This misclassification dilutes epidemiological measures of association, making it more difficult to find significant associations or take regulatory action. In contrast, whole-genome sequencing has demonstrated significant heterogeneity in this particular PFGE subtype [18]. Although the PFGE matching criteria of “indistinguishable” and “different” have been useful, finer gradations of relatedness have proved impractical. Early efforts to develop measures of evolutionary relatedness of patterns [19] were useful in defined outbreaks but not for sporadic case surveillance. This is due to (1) the lack of internal standards against which one could measure relatedness in this setting, (2) uncertainty about the uniqueness of each band which is measured only by size, and (3) issues of transitivity caused by inherent run-to-run variation. It is in the “determination of scope” phase of outbreak investigations [20] where this inability is most important. It is during this phase that investigators try to learn what other cases have a high likelihood of involvement.

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Conclusion Despite its limitations, PFGE has arguably been the most successful tool of molecular epidemiology to date, as measured by actionable public health findings. In foodborne disease surveillance, where PFGE has enjoyed its most visible successes, thousands of PFGE clusters are investigated each year in countries around the world. In this arena the major limitation has been in our ability to obtain quality exposure data, not in the laboratory method. We expect that lessons learned from this remarkable technology will be important into the foreseeable future.

References 1. Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37(1):67–75 2. Van der Ploeg LH, Schwartz DC, Cantor CR, Borst P (1984) Antigenic variation in Trypanosoma brucei analysed by electrophoretic

separation of chromosome-sized DNA molecules. Cell 37(1):77–84 3. Powell NG, Threlfall EJ, Chart H, Rowe B (1994) Subdivision of Salmonella enteritidis PT 4 by pulsed-field gel electrophoresis: potential for epidemiological surveillance. FEMS Microbiol Lett 119(1–2):193–198

Pulsed-Field Gel Electrophoresis for Disease Monitoring and Control 4. Thong KL, Cheong YM, Puthucheary S, Koh CL, Pang T (1994) Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis. J Clin Microbiol 32(5):1135–1141 5. Beall B, Cassiday PK, Sanden GN (1995) Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis. J Clin Microbiol 33(12):3083–3086 6. Stewart PR, el-Adhami W, Inglis B, Franklin JC (1993) Analysis of an outbreak of variably methicillin-resistant Staphylococcus aureus with chromosomal RFLPs and mec region probes. J Med Microbiol 38(4):270–277 7. Hanifah YA, Hiramatsu K (1994) Pulsed-field gel electrophoresis of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections. Malays J Pathol 16(2):151–156 8. Hennessy TW, Hedberg CW, Slutsker L, White KE, Besser-Wiek JM, Moen ME, Feldman J, Coleman WW, Edmonson LM, MacDonald KL, Osterholm MT (1996) A national outbreak of Salmonella enteritidis infections from ice cream. N Engl J Med 334(20):1281–1286 9. Jacobson JA, Chester TJ, Fraser DW (1997) An epidemic of disease due to serogroup B Neisseria meningitidis in Alabama: report of an investigation and community-wide prophylaxis with a sulfonamide. J Infect Dis 136(1): 104–108 10. Green MJ, Cawley PF (1980) Antimicrobial susceptibility of Neisseria meningitidis isolated from 1975-1979. N Z Med J 92(672): 380–382 11. Bender JB, Hedberg CW, Besser JM, Boxrud DJ, MacDonald KL, Osterholm MT (1997) Surveillance by molecular subtype for Escherichia coli O157:H7 infections in Minnesota by molecular subtyping. N Engl J Med 337(6):388–394 12. Bender JB, Hedberg CW, Boxrud DJ, Besser JM, Wicklund JH, MacDonald KL, Osterholm MT (2001) Molecular subtype surveillance of Salmonella typhimurium, Minnesota, 19941998. NEJM 344:189–195 13. Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV, CDC PulseNet Task Force (2001) PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis 7(3): 382–389

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14. McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC (2003) Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 41(11): 5113–5120 15. Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M, Zinn CE, Fussing V, Salmenlinna S, Vuopio-Varkila J, El Solh N, Cuny C, Witte W, Tassios PT, Legakis N, van Leeuwen W, van Belkum A, Vindel A, Laconcha I, Garaizar J, Haeggman S, Olsson-Liljequist B, Ransjo U, Coombes G, Cookson B (2003) Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol 41(4):1574–1585. doi:10.1128/ JCM. 41.4.1574-1585.2003 16. Rainbow J, Jewell B, Danila RN, Boxrud D, Beall B, Van Beneden C, Lynfield R (2008) Invasive group a streptococcal disease in nursing homes, Minnesota, 1995-2006. Emerg Infect Dis 14(5):772–777. doi:10.3201/ eid1405.070407 17. Popovic T, Schmink S, Rosenstein NA, Ajello GW, Reeves MW, Plikaytis B, Hunter SB, Ribot EM, Boxrud D, Tondella ML, Kim C, Noble C, Mothershed E, Besser J, Perkins BA (2001) Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C. J Clin Microbiol 39(1): 75–85 18. Allard MW, Luo Y, Strain E, Pettengill J, Timme R, Wang C, Li C, Keys CE, Zheng J, Stones R, Wilson MR, Musser SM, Brown EW (2013) On the evolutionary history, population genetics and diversity among isolates of Salmonella Enteritidis PFGE pattern JEGX01.0004. PLoS One 8(1):e55254 19. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33(9):2233–2239 20. Reingold AL (1998) Outbreak investigations— a perspective. Emerg Infect Dis 4:21–27

Chapter 2 Harmonization of PFGE Profile Analysis by Using Bioinformatics Tools: Example of the Listeria monocytogenes European Union Reference Laboratory Network Benjamin Félix, Sophie Roussel, and Bruno Pot Abstract Standardization in comparison and interpretation of profiles is an integral part of the analysis of gels from pulsed-field gel electrophoresis. Using Listeria monocytogenes as an example, this chapter outlines the analytical process using the software BioNumerics. Keywords BioNumerics, Listeria, Profile analysis

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Introduction In Europe, listeriosis caused by Listeria monocytogenes, continues to be a serious food-borne illness, where 1,642 confirmed human cases of listeriosis were reported in 2012, including 198 deaths [1]. Molecular typing of this bacterium is essential for surveillance purposes. Pulsed-field gel electrophoresis (PFGE) is invaluable for establishing epidemiological links during routine surveillance and outbreak investigations. The PFGE-based surveillance network, PulseNet, has been used in the USA and in Canada and the use of databases, including food and clinical isolates, has facilitated the detection of outbreaks on numerous occasions [2–6]. Although whole-genome sequencing shows great promise for L. monocytogenes typing in the future, it is not currently widely available. While PulseNet Europe was established in 2003 [7], it later ceased activities in 2006 due to a lack of funding [8]. However, since then efficient networks have been set up and they cooperate closely to improve the exchange of information and molecular typing. The European Centre for Disease Prevention and Control (ECDC) coordinates a network of national public health

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_2, © Springer Science+Business Media New York 2015

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laboratories (NPHLs), in charge of typing L. monocytogenes strains isolated from national clinical cases. In 2012, the ECDC developed a pilot Molecular Surveillance System (MSS) as part of the epidemiological surveillance system (TESSy). This molecular surveillance database has the objective to share, in real time, the molecular epidemiological information and PFGE data on strains isolated from human cases [9]. ANSES Maisons-Alfort Laboratory for Food Safety has been designated as the EU Reference Laboratory (EURL) for L. monocytogenes by DG SANCO of the European Commission. It coordinates a network of 37 National Reference Laboratories (NRLs) in 29 Member States (MSs) and Norway [10, 11]. Most of these NRLs work on typing strains of food, environmental and veterinary origin. The EURL activities of the past few years (annual workshops, training sessions, proficiency testing trials) have enabled NRLs to reinforce and consolidate their typing capabilities [13, 14]. One of the EURL tasks is to harmonize L. monocytogenes typing throughout Europe. For this reason, the EURL has developed standard operating procedures (SOPs) for serotyping and PFGE that are shared with all the NRLs [13]. Moreover, the EURL has developed an SOP for PFGE profile interpretation that reduces the operatordependent subjectivity [12]. These SOPs are based on BioNumerics tools (Applied Maths, Saint-Martens-Latem, Belgium). There was no European molecular database for centralizing and sharing molecular data obtained from food strains (as distinct from TESSy, the clinical strain database). Therefore, in 2012, the EURL set up a L. monocytogenes database that includes typing results (serotyping and PFGE) as well as epidemiological information on strains isolated from food, environmental or animal samples. That database, known as the “EURL L. monocytogenes Database” (EURL Lm DB), is shared within the NRL network. Its purpose is to gather and compare PFGE profiles in the European food chain [14, 15]. The EURL Lm DB is currently effective and is successfully used in European surveillance, in combination with the human strain databases. The EURL Lm DB architecture and the underlying bioinformatics tools developed were specifically designed for this project, in close collaboration between Applied Maths and the EURL. The EURL Lm DB is hosted by the EURL. The network is based on a machine-to-machine communication over the Internet using the software BioNumerics. It was based on PulseNet USA communication scripts, but also included innovative bioinformatics functionalities recently developed by Applied Maths (Fig. 1). The objective was to develop a harmonized approach to analysis of data flowing within the L. monocytogenes European NRLs/ EURL database network. In the first part, we demonstrate in detail the bioinformatics tools, in particular the fingerprint analysis/database management tools. In the second part, we show how their

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EURL database

Direct transmission to EURL database without curation at national level

Curation at EURL level; transmission of comments and official pulsotype returned for synchronization with the national level.

National database Synchronization with central national database. Submission of profiles for centralization at national level

Local laboratories

Fig. 1 Information workflow within the synchronization network

regular use first of all made the process of interpretation PFGE profile easier and secondly allowed NRL/EURL network to exchange and share PFGE profiles.

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Bioinformatics Tools Developed by Applied Maths

2.1 The Software Requirements

In order to compare PFGE profiles between different laboratories, it is important to respect rigorously a very high level of standardization, both in terms of electrophoresis conditions as well as in terms of data normalization and data analysis. In order to be able to compare strains on a longer term, the construction and maintenance of a database with advanced querying possibilities is indispensable. The value of the database will depend on the reproducibility (standardization) of the experimental data stored and on the quality of the documentation and adjunct information. Strain documentation (meta data such as name and number of the sample, date of isolation and date of analysis, geographical information, source of isolation, etc.) as well as additional microbiological information (resistance and virulence profile, phenotype information, sequence information, etc.) are indispensable in the process of an outbreak detection. When exchange of data between multiple reference centers is required, it will be important to be able to exchange data in a structured and confidential way, without the need to renormalize or change data file formats. The BioNumerics™ software discussed here has been built to accommodate all the above requirements.

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The software will not cover, however, the functionality linked to an outbreak investigation, which involves the collection and statistical analysis of epidemiological data once an outbreak has been detected. 2.2 The Software Architecture

The BioNumerics™ software has been designed as a general biological databasing and data analysis and visualization tool. The database is driven by ODBC (open database connectivity) linking to a variety of relational database engines (Oracle®, Microsoft SQLServer®, MySQL, PostgreSQL, etc.). The basic entity is an entry, defined at a specific level in the database. Levels can be used to structure the relational database, reflecting the hierarchy of the information to be stored (Fig. 2). Each entry can be matched to a number of information fields that can be unique per level, or shared between different levels. An overview level allows all levels to be visualized in a single window. Information fields can be added at any time. The database is object oriented, has full user management and is equipped for integration in an FDA 21 CFR Part 11 compliant environment (electronic record keeping). The “functional” level will usually be a biological sample: an individual living organism (animal, plant, bacterium, yeast, fungus, etc.) or a more complex sample (food, soil, feces, etc.), for which experimental and metadata are collected. In the case of surveillance of microorganisms, an entry usually matches a bacterial strain or Operational Taxonomic Unit (OTU), characterized by a unique identifier, called “key.” The latter can be automatically assigned by the software or chosen by a user-defined strategy. Entries can be sorted by any information field, and a variety of search functions allows the selection of entries to be made and stored as subsets. A selection of entries is the basis for any analysis, executed in a so-called “comparison window,” offering imaging, alignment, cluster, phylogeny, and principal component analysis, polymorphism analysis, statistical analysis, identification, etc.

Fig. 2 The database design window showing the user-specific architecture of each database. Each vertical column represents a database level. Each level can have unique or shared information fields

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The software has been equipped with a large number of “plugins,” offering specific functionality for, for example, import of the variety of files from a wide diversity of hardware and software products used in biological experimentation, for linking to specific web services (services for multilocus sequence typing (MLST), Staphylococcus protein A (SPA), mycobacterial interspersed repetitive units-variable number tandem repeats (MIRUVNTR) typing) and public or private databases (NCBI, EMBL, …), or for managing data export (HIV resistance plugin) and exchange (PulseNet communication). The user can develop and use Python scripts from within the software to further interact with the software at almost any level. In order to cope with the diversity of experimental data types, so-called “experiment type” containers will need to be created [16]. In total seven different experiment types are available: 1. The Fingerprint experiment type, accommodating fingerprints or results of fragment analysis from gels (photographs and scans) or from capillary sequencers or capillary bio-analyzers, and spectra from Maldi, HPLC, or gas chromatography equipment. 2. The Character experiment type will accommodate any binary, categorical or numerical data set, ranging from MLST types, FAME, antibiotic resistance levels, SPA- and Spoligo-types to high-throughput microarray and gene chip data as well as whole-genome SNP and MLST data. 3. The Sequence type will store and manage sequences from DNA and amino acids, including Sanger DNA sequences as well as Next Generation Sequences (NGS) from genome or metagenomics analysis. 4. The Short Read Set experiments will manage the short reads typically generated by NGS technologies and will automatically prepare K-mers of length 7 for quick sequence comparison. 5. The Optical Map type will store the maps generated by the OpGen® technology. 6. Trend data, like growth curves or qPCR experimental data, will be managed by the Trend data experiment type, and finally, 7. The Matrix data type will store full matrix data (similarity matrices from other software) or partial matrices, such as the results of DNA–DNA hybridization experiments. Within each of these experimental groups, the user can create custom types of experiments, reflecting the nature of the data collected. Analysis of one experiment type may lead to the (automatic) supplementation of another experiment type: analysis of Maldi peaks may lead to a species-specific peak-set stored in a character data type; comparison of a chromosomal sequence to a reference sequence may lead to a SNP set stored in a character type as well.

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In the frame of this chapter we will exclusively focus on the Fingerprints experiment type. The importance of the other experiment types should be seen in the validation or support of PFGE based surveillance or outbreak conclusions, by using additional epidemiological and/or typing information stored in the database. The use of this additional information has been facilitated by the possibility to link different data sets (experiment types) to a single biological sample and to allow combined visualization and analysis in one single window. 2.3 The Fingerprint Experiment Type

Fingerprints are defined as experiments that lead to densitometric records seen as peak or band profiles, and for which nothing else is known other than the running distance (Rd) or a metric unit such as molecular weight, fragment length, or isoelectric point. Examples are electrophoresis patterns obtained from images, or spectrophotometric curves from dedicated hardware (capillary sequencers, bio-analyzers). Pulsed Field Gel Electrophoresis (PFGE) profiles will be analyzed and stored in the fingerprint experiment type, along with its specific settings such as reference marker used for the normalization of the profile, the MW regression (link between distance and length or molecular weight), the stain specifications, proposed band matching tolerance, advised similarity coefficient, preferred clustering method, etc. Since electrophoresis is an important component for studying relationships in biology, comprehensive tools for preprocessing electrophoresis fingerprints have been incorporated in BioNumerics. These tools include reading different graphical file formats, lane-finding, normalization (alignment of patterns), band finding and quantification, band matching, etc. BioNumerics possesses advanced “remapping” functions which allow gels normalized to different molecular weight markers or reference bands to be automatically remapped into each other in real time. This feature is of particular interest for institutions that exchange fingerprints in a collaborative research or surveillance context.

2.4 The Data Analysis Functionality

Analysis happens on queried selections from the database, included in a comparison window. The purpose of the analysis in most cases is to calculate similarities between entries with the purpose of identifying unknown entries or to group larger datasets to reveal clusters or phylogenetic relationships. BioNumerics offers two main types of mathematical comparison methods: (1) Cluster Analysis, revealing a hierarchical grouping or a phylogeny, and (2) Principal Component Analysis (PCA), Multi-Dimensional Scaling (MDS), or Self Organizing Maps (SOM), resulting in a visual organization of potential groups of entries. A third type of analysis methods is not intended to be used on entries but to compare different characterization techniques and to calculate concordance between them.

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2.4.1 Cluster Analysis

A dendrogram is still the most generally used grouping method to represent relationships between organisms. It requires the calculation of a (dis)similarity matrix, using a (dis)similarity coefficient. For the comparison of PFGE profiles two types of coefficient can be used: the Pearson product–moment correlation, using the entire profile, or the binary Jaccard or Dice coefficients, scoring similarities between band positions. The latter coefficients are used almost exclusively for PFGE patterns [17]. While (dis)similarity matrices contain valuable information, revealing similarities between each individual pair of entries, the matrix is not structured and interpretation is difficult, especially for larger data sets. Therefore, the matrix needs to be reordered, revealing highly similar sets of entries and illustrating similarities between different clusters. This is achieved through clustering. Among the rooted tree algorithms, BioNumerics offers the Unweighted Pair Grouping Method using Arithmetic averages (UPGMA), single (nearest neighbor) linkage, complete (furthest neighbor) linkage, and the “Ward” method. The Neighbor Joining method can also be used to construct unrooted trees. As information is generally lost during clustering, methods are necessary to evaluate the confidence level of a tree or of each individual branch in the tree. Frequently, the standard deviation or the cophenetic correlation is used, calculated at each branching level and the root.

2.4.2 PCA, MDS, and SOM

While not used as frequently as dendrograms, Principal Component Analysis (PCA) is a very attractive and faithful alternative to discriminate small numbers of groups or closely related organisms. Native PCA can only be applied directly on complete datasets containing the same characters and will, therefore, not need a similarity matrix to be calculated. To this extent, the comparison of PFGE patterns will require a band matching table to be prepared for the set of entries analyzed. A PCA results in a two or three-dimensional representation in which the entries are scattered more closely or distantly according to their relationship. Real-time rotation of the 3D-presentation modes enhances the spatial perception and visualization of more complex structures. In BioNumerics the spatially scattered entries can be connected using the branching order of a corresponding dendrogram, obtained on the same dataset, improving the understanding of the obtained groups. In contrast to PCA, Multi-Dimensional Scaling (MDS) does not start from the raw data set as input, but requires a matrix of similarity values instead. This is an advantage over PCA which makes the method more generally applicable. The result is a threedimensional structure with the same features as a 3D PCA plot. In BioNumerics, the classical MDS has been extended with an optional distance optimization algorithm, which iteratively optimizes the distances between the OTU’s in the MDS space according to the similarity values of the matrix. All this makes the method an

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excellent tool for discriminating closely related taxa. Finally, the Self Organizing Map (SOM, also called Kohonen map) is a kind of neural network that classifies entries in a two-dimensional space (map) according to their similarity. Unlike PCA, the distance between entries on the map is not in proportion to the taxonomic distance between the OTU’s. Rather, a SOM contains areas of high distance and areas of high similarity. Such areas can be visualized by different shading, for example when a darker shading is used in proportion to the distance. The grouping technique resembles the training of a neural network and is, therefore, completely different from all previously described methods. When a similarity matrix is considered as the character set, a SOM can be applied to similarity matrices, which makes the technique also suitable for grouping of electrophoresis patterns that are compared pair by pair using a band matching coefficient such as Dice. SOMs provide an interesting alternative to conventional grouping methods and are an extremely useful tool for the analysis of very large data sets with a very large diversity level. 2.4.3 Analysis of Congruence Between Data Sets

Besides the data analysis features mentioned above, BioNumerics also has the ability to create composite data sets of any type, combining two PFGE profiles, for example, obtained with different restriction enzymes, or combining PFGE data with MLST data. The software has the mathematical methods to create global comparisons based upon the combination of these different data sets. It is also possible, however, to evaluate the outcome of a cluster analysis, for example, on both data sets separately. This can be done on the dendrogram level, drawing consensus dendrograms, or on the (dis)similarity matrix level, calculating the concordance between different characterization techniques or calculate a clustering of congruence between the different techniques used. This is especially interesting when more than two techniques are compared, as it allows the user to group techniques that separate the data set in a similar way, or to find which technique is the closest to the consensus classification, since this technique/restriction enzyme will be generally be the most reliable for the organisms under study. In this approach, the software will calculate a “clustering of clusterings,” or in fact, a matrix of matrices. Matrices are compared in a pairwise manner by comparing corresponding similarity values by either Kendall’s Tau coefficient or by the product– moment correlation. This results in a new matrix, expressing the global similarity or congruence between different techniques. This matrix in turn can be clustered into a dendrogram, and congruent techniques can be visualized at a glance. Pairwise comparisons between any two techniques are obtained by plotting the corresponding similarity values on an X–Y diagram, and by calculating a regression. Such regressions are very useful both to visualize the

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congruence between techniques and to reveal the taxonomic level or depth of a technique compared to another: it shows whether one technique/restriction enzyme is discriminative at a lower or higher level than another technique/restriction enzyme and provides insight in the limitations and benefits of each technique/ restriction enzyme in building identification strategies or validating epidemiological findings. 2.5 The Data Communication Functionality

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In the above mentioned PulseNet networks, exchange of information among different reference laboratories is of the utmost importance to detect large scale outbreaks. BioNumerics offers standardized ways to perform this exchange. The earlier component behind the exchange concept, still in use, is the so-called “bundle,” a general and open standard for the exchange of database information among different partners. A bundle contains a set of database entries, ranging from one single entry to several thousands, and for each entry one or more experiments and information fields, all defined by the user. If an imported bundle from another collaborating reference laboratory is opened, any additional database fields and experiment types that are not already defined in the database of the recipient database are automatically added to the database. Alternatively, if the same database field or experiment has been given a different name by the sender, the receiver may rename any database field or experiment to match the name of the own database fields or experiments. A second way for data exchange is through the current XML standard. Similarly as for bundles, the user can define the context and content of an XML file, which can be send to a recipient by the software or through e-mail. Bundles and XML files exchanged between different copies of the software are generally encrypted to prevent unauthorized access to the information.

Analyzing PFGE Data The data processing of a fingerprint type such as a PFGE gel image comprises four steps, briefly discussed below.

3.1 Image Import and Preprocessing

A TIFF, GIF, JPEG, or PNG file can be imported through the import wizard, allowing to crop the image to the relevant size and to rotate the image if necessary. The wizard will also make sure the image is assigned to the right experiment type or to create a new one if necessary (e.g., a new restriction enzyme in a PFGE experiment). When the image is known to the software it can be visualized in the gel analysis window and the lane finding algorithm can be run (Fig. 3). This will define the gel strips, to be excised from the image and stored in the database with the

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Fig. 3 Lane finding in the active area of the gel

respective entry. As gels strips will also be the basis for the calculation of the intensity profiles (Subheading 3.2), which will be used for normalization (Subheading 3.3) and band detection (Subheading 3.4), they should be prepared with care and preferably cover the active area of the PFGE profile, i.e., the area covered by the marker lanes and marker bands. Gel strips can be found automatically, but correction for displacements, differences in width or small distortions might be necessary (Fig. 3). Image brightness and contrast can be adapted to allow an optimal visualization for further processing. 3.2 Defining Densitometric Curves

In this step the gel strips extracted from the image file will be automatically used to define the densitometric curves for each lane in the image. This process will involve the averaging of intensities in an active zone defined by the user. The broader the linear zone, the less sensitive the densitometric curve will be to artifacts. Different filtering, smoothing, and averaging methods can be used to obtain an even more reliable curve (Fig. 4).

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Fig. 4 Calculation of densitometric curves from gel strips. The zone defined by the red lines is the active zone used for averaging intensities. The resulting profile on the right can be further filtered and smoothed

3.3 Curve Normalization

In this step the reference pattern position or positions will need to be identified. Reference lanes will be used to compare to the reference pattern stored in the respective fingerprint experiment type of the database (called “Standard”). Displacements between reference bands detected on the reference patterns and reference bands on the Standard will be corrected by compression or extension of the curve. Neighboring data lanes will also be corrected. Color grades (pale to dark) will indicate the degree of distortion for easy evaluation of possible misalignments between the marker lanes and the Standard (Figs. 5 and 6).

3.4

The normalized curves are subjected to a band filtering algorithm in this step. A minimum profiling level will need to be identified, specifying the minimum degree of elevation with respect to the background (in percent) for a “true” band to be recognized. For PFGE profiles, often a “gray zone” is defined, specifying bands that will be marked as uncertain in the database (Figs. 7 and 8).

Defining Bands

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Fig. 5 Illustration of the “distortion bar” functionality which enables detection of inappropriate positioning of the reference bands or abnormal migration of the reference system

Fig. 6 A gel in the normalization process. The Standard on the left is used to match the gel being analyzed. Reference marker bands, labeled in green, on reference marker lanes have been used to match the gel to the internal Standard. Neighboring lanes have also been corrected and distortions introduced are indicated by pale colors (yellow and blue). Normalized curves can now be stored in the database and used for analysis using, for example, the Pearson correlation coefficient. In case a band-based coefficient needs to be used, individual bands will need to be identified

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Fig. 7 The band search window with a minimum profiling of 10 % and a gray zone of 5 %

Fig. 8 Resulting band assignment. Full green lines indicate certain bands, green oval symbols indicate uncertain bands

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4 The Use of Bioinformatics Tools in the EURL SOP for PFGE Profile Interpretation The EURL SOP provides guidance for the NRLs, to analyze a PFGE profile in their local database, using the four steps process explained above. The SOPs point out the most critical steps and how to judge them [12, 14]. For each of these steps, adapted bioinformatics tools may help the users in their task. 4.1 Bioinformatics Support Tools for the Visual Interpretation of Gels and Their Profiles

This first part describes how to frame, process, and assign markers on the profiles generated from PFGE, within a fingerprint experiment type. The profile has to be framed entirely and should not contain background or debris which impedes interpretation of the image be occulted on the gel. The tools for gel framing, available at the “strip” step (Subheading 3.1) in BioNumerics, facilitate definition of the profile analysis area. The profiles are then transposed into a densitometric curve. At the “curve” step (Subheading 3.2), the densitometric curve extraction area is defined manually. This avoids minor spots that could impede interpretation of the image by twisting the extraction strip. The SOP requires that the densitometric curve extraction area does not exceed one third of the profiles width. The densitometric curve calculation enables better visualization of the profile for easier quality assessment of: (1) peak intensity shape, (2) peak definition and (3) background intensity level. No mandatory parameters are established at this step. However, the optimal parameters proposed by the “spectral analysis” functionality are recommended to adapt the image processing to each laboratory image quality. For L. monocytogenes, the Standard used for the normalization of the profile is the international standard reference system Salmonella Braenderup H9812 XbaI restriction profile [18]. In the EURL SOP, the band size is used to define the profile interpretation analysis framework between 1,135 and 33 kbp. At this step, the reference bands on the gel image have to be marked with accuracy, at the average position of every signal (Fig. 9). The software helps the user by placing automatically the markers on the signal. At this step (Subheading 3.3), an accurate band marking is crucial. When all reference lanes are assigned and marked, the normalization of the gel is performed. The software will correct the profile size and introduce a calculated distortion in order to align the bands of the reference system from the current gel to the Standard. The migration in the gel should not be distorted excessively in comparison to the Standard associated with the experiment normalization. The distortion evaluation by the so-called “Distortion bars” functionality, enables the visualization of the distortions, shown as colored bars. Light colors (sky blue or yellow) indicate little or no distortion with respect to the Standard. Darker colors (red or bright blue) indicate a stronger distortion which may be

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Fig. 9 The reference system Salmonella Braenderup H9812 XbaI profile used for the normalization of a Lm PFGE gel in BioNumerics. The internal reference system marked on the left side gives the link between migration distance and DNA fragment molecular weight. The positions of the reference bands detected in the reference lane in the center of the gel are automatically detected and marked with green indicators. Displacements to the expected normalized position are shown by small green arrows. Following the EURL SOP, Salmonella Braenderup H9812 XbaI profile is marked until the 33 kbp band. The representation at the right is the curve’s intensity plot

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Fig. 10 Example of eventual correction of the automatic band detection results by using the densitometric curve of the profile. The blue arrows show how the band markers are a help to determine the average position of each signal

compensated by the software (Fig. 5). Black coloring indicates distortions which are too large to be counteracted by the software. Profiles carrying black color distortion are not analyzed. Black colors may be either indicative of a wrong reference band assignment or will indicate that the gel is not conform the required Standard and should be rerun. After normalization, all band positions of the profile will be associated (“bands” Subheading 3.4) with a DNA fragment size based on representing the link between fragment size and migration distance. Every DNA fragment signal on the profiles, corresponding to the fluorescent intensity recorded by the camera on the gel from DNA fragments stained with ethidium bromide or equivalent DNA dye, has to be marked with band markers. The fragment sizes have to be determined at the average position of each signal (Fig. 10). This process can be assisted by the band filtering algorithm which is useful to place the markers on the profiles with accuracy. For signals showing irregular shapes (e.g.: shoulder, double peak), the accurate marker position is defined manually according to the signal overlap. The SOP for PFGE profile interpretation includes a validation of the profiles based on the assessment of the band signal intensity [12]. These data are available, from a “peak table” that gives the band height on the densitometric curve. These data are used for automatic validation of the profiles.

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4.2 Bioinformatics Support Tools for Profile Nomenclature Assignment

This second part describes how to interpret, homogenize and finally assign a nomenclature to a pulsotype in the NRLs database. All profiles available in the EURL Lm DB can be compared with local NRL profiles. The EURL Lm DB can thus be considered as a routine assessment tool for NRLs. The process starts with fast band matching comparison of the NRL database profiles with the EURL Lm DB profiles. A tolerance of 1 % is recommended in the EURL SOP to perform the matching. From the matching profiles found, a selection limited to closely related profiles is made. This selection can be downloaded from the EURL Lm DB to the NRL database. The downloaded profiles also called “bundles” (Subheading 2.5) are available for profile comparison until the active session of the software is closed. Subsequently, the downloaded profiles are compared to the local profiles and interpreted within a “comparison file.” The assignment of the bands on the profile is made by comparison of the local profile with the EURL Lm DB profiles, which are taken as reference for interpretation. The operator has to (a) verify the homogeneity of their own profiles with those of the downloaded bundles, (b) change the local profiles to match with reference profiles as much as possible, (c) check that a band is always placed on a true signal. This step is made possible by, (1) the direct display of densitometric curves on the profiles within a comparison, (2) the “band matching” functionality that allows direct modification of the profile within a comparison, and (3) the “pairwise comparison” which allows a profile-to-profile parallel display for a final profile comparison.

4.3 Bioinformatics Support Tools Used in the EURL Lm DB for Curation

The profiles submitted to the EURL Lm DB by the NRLs are already interpreted by the NRL according to the EURL SOP. The curation task is to (1) validate the profile quality assessment, (2) include the newly submitted profiles within the database, (3) assign an official pulsotype number, and (4) make these profiles available for all the network users. This process is complex because it gathers profiles from several NRLs and requires adapted bioinformatics tools for database consistency management: identification project tool and comparison file color marking. The curator uses the whole database as a reference to assign pulsotype numbers. This strategy aims to assign each profile to a group of profiles similar at 90 % (“identification group”) and then assign the profile to a pulsotype within this group. The curator task is to minimize the diversity within an identification group by reducing artificial diversity generated by the operator’s interpretation of the profile. The first step of the interpretation starts by the comparison of the new profile against all identification groups. This comparison is made automatically using the “identification project” tool. This tool compares the new profiles with all the identification group profiles in the database. It provides a matching score given as the mean similarity between the new profile and the

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components of every “identification group.” The new profile is then included within the identification group with the “nearest mean similarity.” At this stage the operator has to respect the following identification group definition: (a) verify the homogeneity of the new profile with the identification group content, (b) change the new profile to match with its assigned identification group as much as possible, (c) perform profile modification within the identification group limit (90 % similarity within identification group components), (d) check that a band is always placed on a true signal [12]. At the end of the process spanning between identification groups is checked within a global dendrogram, gathering all the profiles of the database. In this dendrogram the identification groups are mapped according to a color code. Overlapping is detected when the color groups are mixed up. 4.4 Bioinformatics Support Tools for EURL and NRL Database Synchronization

The curation process implies that the interpretation of the same profile will be changed between the NRLs’ database and EURL Lm DB. After curation, the synchronization between the NRL and the EURL databases allows homogenous interpretation of the profiles in the network. The EURL Lm database was designed to allow synchronization with the NRL databases [15]. This synchronization is possible and requires NRLs to update their profiles. The synchronization is made through a feedback of the curated profiles into the NRL local database. This transmission of information is made through XML file format (Subheading 2.5). The information transmitted during the synchronization process are: the image processing parameters, the reference system band position, the profile band positions, the pulsotypes (into a database field), curator comments (into a database field) and profile status (into a database field).

4.5 Bioinformatics Support Tools for Traceability of the Curator Work

The synchronization functionality also provides information on the curator activities: which profiles were modified, when the modification occurred and what was modified. This information is available in the NRL database via an “Event log” dialogue box. According to the information provided, the NRL can identify and synchronize the profiles modified in the EURL Lm DB since its last update and then benefit from the last changes made by the curator.

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Conclusion: Prospects The EURL has developed SOPs for PFGE and for PFGE profile interpretation. Moreover, the EURL has set up a European molecular typing database, to exchange typing data within the NRL/ EURL network. The two SOPs and the database were built on

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bioinformatics tools available in the software BioNumerics. The supportive functionalities proposed by the EUR Lm DB are: 1. To provide valuable information for epidemiological investigation in case of an outbreak (serotype, food matrix, sampling date and profile frequency in the EURL Lm database). 2. To allow the implementation of the curator validated profiles in the NRLs’ database by synchronization. This implementation gives the opportunity for the NRLs to establish at national level a network with partner laboratories (Fig. 1). 3. To outsource to the EURL the interpretation of the NRL profiles. This process (1) enlightens the NRL local work on PFGE profiles and (2) allows the NRLs to benefit from a common and central interpretation of their own profiles with the whole network. Compared to other PFGE database projects such as PulseNet USA [19], TESSy MSS [9], Food Microbe Tracker [20], the synchronization functionality is innovative. Using the EURL Lm DB has encouraged the NRLs to widely use their national databases and to set-up a network of national partners involved in the national surveillance. The EURL Lm DB was proposed as an innovative project created by the NRLs/EURL network in close collaboration with Applied Maths and supported by a grant from the European Commission’s Directorate General (DG SANCO). The steering committee comprises eight NRL representatives, EFSA and ECDC. This project successfully contributed to the development of the forthcoming EFSA food, environment and animal molecular typing database for the main food-borne pathogens. In 2015 the EURL Lm DB content will be transferred into the L. monocytogenes EFSA database. The EURL will be in charge of EFSA database curation [21]. References 1. European Food Safety Authority (2014) The European Union Summary Report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA J 12(2): 3547, 1–312 2. Cdc U (2010) Outbreak of invasive listeriosis associated with the consumption of hog head cheese–Louisiana, 2010. MMWR Morb Mortal Wkly Rep 60:401–405 3. Cdc U (2011) Multistate outbreak of listeriosis associated with Jensen Farms cantaloupe–United States, August-September 2011. MMWR Morb Mortal Wkly Rep 60:1357–1358 4. Choi MJ, Jackson KA, Medu C, Beal J, Rigdon CE, Cloyd TC et al (2014) Notes from the field: multistate outbreak of listeriosis linked to

soft-ripened cheese - United States, 2013. MMWR Morb Mortal Wkly Rep 63:294–295 5. Gaulin C, Gravel G, Bekal S, Currie A, Ramsay D, Roy S (2014) Challenges in listeriosis cluster and outbreak investigations, Province of Quebec, 1997-2011. Foodborne Pathog Dis 11:1–7 6. Gilmour MW, Graham M, Van Domselaar G, Tyler S, Kent H, Trout-Yakel KM et al (2010) High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak. BMC Genomics 11:120 7. Martin P, Jacquet C, Goulet V, Vaillant V, De Valk H (2006) Pulsed-field gel electrophoresis of Listeria monocytogenes strains: the PulseNet

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Benjamin Félix et al. Europe Feasibility Study. Foodborne Pathog Dis 3:303–308 Swaminathan B, Gerner-Smidt P, Ng LK, Lukinmaa S, Kam KM, Rolando S, Gutierrez EP, Binsztein N (2006) Building PulseNet International: an interconnected system of laboratory networks to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging foodborne diseases. Foodborne Pathog Dis 3:36–50 van Walle I (2013) ECDC starts pilot phase for collection of molecular typing data. Euro Surveill 18 EURL-Lm (2014). Accessed November 15, 2014. Available from: https://eurl-listeria. anses.fr/ Lombard B (2012) Reliability of measurement results in food microbiology: the contribution of reference laboratories in the European Union and of international/European standardization. Accred Qual Assur 17:223–229 Félix B, Brisabois A, Dao TT, Lombard B, Asséré A, Roussel S (2012) The use of pulsed field gel electrophoresis in Listeria monocytogenes sub-typing: harmonization at the European Union level. In: Magdeldin S (ed) Gel electrophoresis: principles and basics, vol 1, 14th edn. INTECH, Rijeka, Croatia, pp 241–254 Felix B, Dao TT, Grout J, Lombard B, Assere A, Brisabois A, Roussel S (2012) Pulsed-field gel electrophoresis, conventional, and molecular serotyping of Listeria monocytogenes from food proficiency testing trials toward an harmonization of subtyping at European level. Foodborne Pathog Dis 9:719–726 Felix B, Niskanen T, Vingadassalon N, Dao TT, Assere A, Lombard B, Brisabois A, Roussel S (2013) Pulsed-field gel electrophoresis proficiency testing trials: toward European harmonization of the typing of food and clinical strains of Listeria monocytogenes. Foodborne Pathog Dis 10:873–881

15. Felix B, Danan C, Van Walle I, Lailler R, Texier T, Lombard B, Brisabois A, Roussel S (2014) Building a molecular Listeria monocytogenes database to centralize and share PFGE typing data from food, environmental and animal strains throughout Europe. J Microbiol Methods 104:1–8 16. Vauterin L, Vauterin P (2006) Integrated databasing and analysis. In: Stackebrandt E (ed) Molecular identification, systematics, and population structure of prokaryotes. Springer, Berlin, pp 141–217 17. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33(9):2233–2239 18. Hunter SB, Vauterin P, Lambert-Fair MA, Van Duyne MS, Kubota K, Graves L, Wrigley D, Barrett T, Ribot E (2005) Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol 43:1045–1050 19. Gerner-Smidt P, Hise K, Kincaid J, Hunter S, Rolando S, Hyytia-Trees E, Ribot EM, Swaminathan B (2006) PulseNet USA: a fiveyear update. Foodborne Pathog Dis 3:9–19 20. Vangay P, Fugett EB, Sun Q, Wiedmann M (2013) Food microbe tracker: a web-based tool for storage and comparison of food-associated microbes. J Food Prot 76:283–294 21. EFSA (2014) Molecular typing of Listeria monocytogenes strains isolated from food, feed and animals: state of play and standard operating procedures for pulsed field gel electrophoresis (PFGE) typing, profile interpretation and curation. EFSA supporting publication, Dec 2014, pp 1–81

Chapter 3 PFGE as a Tool to Track Listeria monocytogenes in Food Processing Facilities: Case Studies Marion Dalmasso and Kieran Jordan Abstract The use of PFGE to track Listeria monocytogenes strains from the food processing environment to the food is explained in this chapter through two case studies. This illustrates the usefulness of this method to identify putative routes of contamination and persistent strains and to help in the implementation of corrective actions in food processing facilities to control the occurrence of this pathogenic bacterium. Keywords Listeria monocytogenes, PFGE, Case study

1

Introduction Listeriosis, a disease caused by the food-borne pathogenic bacteria Listeria monocytogenes, can affect susceptible populations, such as newborn children, the elderly, and immunocompromised persons, with a mortality rate of 20–30 % [1–3]. Infection with L. monocytogenes is associated with the consumption of a range of ready-to-eat foods such as meat, fish, vegetable, and dairy products [4]. The production of safe food is the responsibility of the food business operator as stipulated by the European Union legislation [5] and is based on food safety systems like the hazard analysis of critical control points (HACCP) system and the implementation of general preventive measures such as good hygiene and manufacturing practices. It has been shown that L. monocytogenes can survive improper cleaning and disinfection processes and that exposure to sublethal concentrations of disinfectants supports the survival and growth of these bacteria [6]. Cross-contamination from the environment to food has been reported [7, 8] and can lead in some cases to listeriosis outbreaks [9, 10]. Consequently, it is of utmost importance to limit and control the occurrence and persistence of

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_3, © Springer Science+Business Media New York 2015

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L. monocytogenes in the food processing environment in order to avoid cross-contamination and to ensure the production of safe food for consumption. Environmental sampling is an effective way to assess hygiene and prevent future contamination events [11]. A stringent L. monocytogenes control program, even in small processing facilities, and measures to prevent and control persistent L. monocytogenes contamination in niches in processing facilities are essential. Pulsed field gel electrophoresis (PFGE) is the “gold-standard” method to characterize and compare L. monocytogenes isolates. PFGE not only allows a greater understanding of population diversity but also enables more accurate identification of possible food contamination routes and thus facilitates the establishment of control measures that are likely to address the problem of cross-contamination of food. The aim of this chapter is to illustrate with two case studies the use of PFGE to track sources of contamination with L. monocytogenes of the food processing environment.

2 Case 1: Cross-Contamination with a Persistent Strain from the Food Processing Environment to the Cheese An Irish raw and pasteurized semisoft rind washed farmhouse cheese had a recurrent problem of contamination with L. monocytogenes. Some cheeses were positive for L. monocytogenes in 1999, 2007, 2008, and 2012. Environment samples in the farmhouse cheese processing facility were taken, and isolates were compared using the PulseNet PFGE protocol for L. monocytogenes [12]. Pulsotypes were also compared to existing pulsotypes of isolates found in the farmhouse cheese processing facility during other sampling campaigns. The same pulsotype was found in all the contaminated cheeses from 1999 to 2012 (Fig. 1). This contamination with the same L. monocytogenes strain indicated a persistent strain as the source of contamination of cheese. When compared to the isolates from the processing facility, the strain isolated from cheese displayed an indistinguishable pulsotype with some isolates of the processing environment (Fig. 1). This persistent pulsotype was found in the processing environment in 1999, 2007, 2011, and from May to September 2012. These results indicated cross-contamination from the processing environment to the cheese with a persistent L. monocytogenes strain. During the autumn 2012, the floor was completely renewed in the processing area. Since then, no cheese contamination with L. monocytogenes has been detected.

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Fig. 1 Dendrogram of PFGE pulsotypes combining ApaI and AscI obtained using Bionumerics version 5.10 software (Applied Maths, Belgium). Band matching was performed using the DICE coefficient. The dendrogram was created using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). FPE food processing environment

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Case 2: Contamination of Cheese from Outside the Processing Environment In 2007, an Irish farmhouse cheese produced in a farmhouse cheese making facility with an associated dairy farm faced a problem of contamination with L. monocytogenes. Environment samples were taken outside and inside the processing facility. Isolates were compared using the PulseNet PFGE protocol for L. monocytogenes [12]. Two other sampling campaigns were also performed in 2010 and 2012. Three groups of pulsotypes, G1 to G3, were identified (Fig. 2). In group G1, the same pulsotype was found in the contaminated cheese in 2007 and in straw present on the dairy farm. This pointed to a possible source of L. monocytogenes contamination as the dairy farm. This was also confirmed by the pulsotypes constituting group G2 in Fig. 2. An isolate from cow manure was indistinguishable by PFGE from an isolate found in a drain inside the processing facility, indicating a potential route of contamination from outside to inside the cheese processing facility. The pulsotype found in the cheese and the straw in 2007 (group G1, Fig. 2) was also found on a door in the processing area and in a cheese in 2012. This indicated the presence of a persistent strain in the processing facility. Isolates from the processing environment in 2007 and 2010 and cheese in 2012 shared the same pulsotype, constituting group G3 on Fig. 2.

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Fig. 2 Dendrogram of PFGE pulsotypes combining ApaI and AscI obtained using Bionumerics version 5.10 software (Applied Maths, Belgium). Band matching was performed using the DICE coefficient. The dendrogram was created using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). FPE food processing environment. The dotted lines indicate the three groups of pulsotypes identified in this case study

4

Discussion These two case studies illustrate the occurrence and persistence of L. monocytogenes in the processing environment with the associated risk of cross-contamination from the external environment to the food product. Case 1 showed the persistence of an L. monocytogenes strain in the food processing environment for more than a decade. L. monocytogenes can persist and even proliferate under adverse environmental conditions such as low pH, high salinity, and low temperature, in particular probably due to its ability to form biofilms; to resist to desiccation, acid, and heat [13]; to survive to increased sublethal concentration of disinfectants; or to resist lethal concentrations [14]. In Case 1, the floor was entirely changed in an attempt to clear the processing environment of the persistent L. monocytogenes contamination. However, in order to prevent the reappearance of L. monocytogenes, this corrective action needs to go along with other measures for maintaining or improving physical barriers between outside and inside the processing area, workflows, and cleaning and disinfection procedures. For example, in a food processing facility with a recurrent problem of contamination with L. monocytogenes, PFGE was used to differentiate isolates and to identify persistent strains over time [15]. Based on these results, advice was given to the food business operator regarding increased cleaning and disinfection practices (including use of peracetic acid as a disinfectant), attention to details in cleaning, and improved workflows in order to limit and prevent the dissemination of L. monocytogenes in the processing environment. This resulted in a drastic decrease in the contamination with L. monocytogenes.

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In Case 2, PFGE allowed to identify the external environment as a source of contamination with L. monocytogenes of the processing environment and eventually of the cheese. Farm environment is a well-known potential source of cross-contamination of milk or of the cheese processing facility [16]. Cattle farms are reservoirs for L. monocytogenes strains linked with human disease [17]. Thus, preventing cross-contamination between dairy production and processing facilities is critical to assuring the microbial safety of farmhouse cheese [18]. Similar routes of contamination from outside to inside the processing facility and finally reaching the final food products have also been described recently [19]. In this study, isolates displaying the same pulsotypes were found in the yard, in a vat, on the floor of the processing area, and in a cheese. Pathogens may access the food processing environment through raw material like raw milk [20] or brine [21]. Staff members, mobile equipment, leaks and openings in buildings (doors, windows), or even pests may also be the source for L. monocytogenes contamination in processing facilities [22].

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Conclusion In these two case studies, PFGE was used to characterize and compare L. monocytogenes isolates from the food processing environment and foods. These examples illustrate the usefulness of PFGE to track the routes of contamination with L. monocytogenes of the food processing environment and to identify persistent strains over time. This method is a powerful tool to help in the control and the reduction of L. monocytogenes occurrence along the food chain for the production of safe food.

References 1. Lorber B (1997) Listeriosis. Clin Infect Dis 24:1–9 2. McLauchlin J, Mitchell RT, Smerdon WJ, Jewell K (2004) Listeria monocytogenes and listeriosis: a review of hazard characterisation for use in microbiological risk assessment of foods. Int J Food Microbiol 92:15–33 3. Sleator RD, Watson D, Hill C, Gahan CG (2009) The interaction between Listeria monocytogenes and the host gastrointestinal tract. Microbiology 155:2463–2475 4. Lianou A, Sofos JN (2007) A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J Food Prot 70: 2172–2198

5. European Commission (2005) Commission regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union L338: 1–26 6. Pricope-Ciolacu L, Nicolau AI, Wagner M, Rychli K (2013) The effect of milk components and storage conditions on the virulence of Listeria monocytogenes as determined by a Caco-2 cell assay. Int J Food Microbiol 166: 59–64 7. Lin CM, Takeuchi K, Zhang L, Dohm CB, Meyer JD, Hall PA et al (2006) Crosscontamination between processing equipment and deli meats by Listeria monocytogenes. J Food Prot 69:71–79

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8. Nakama A, Matsuda M, Itoh T, Kaneuchi C (1998) Molecular typing of Listeria monocytogenes isolated in Japan by pulsed-field gel electrophoresis. J Vet Med Sci 60:749–752 9. Gaulin C, Ramsay D, Bekal S (2012) Widespread listeriosis outbreak attributable to pasteurized cheese, which led to extensive cross-contamination affecting cheese retailers, Quebec, Canada, 2008. J Food Prot 75:71–78 10. Winter CH, Brockmann SO, Sonnentag SR, Schaupp T, Prager R, Hof H et al (2009) Prolonged hospital and community-based listeriosis outbreak caused by ready-to-eat scalded sausages. J Hosp Infect 73:121–128 11. Tompkin RB (2002) Control of Listeria monocytogenes in the food-processing environment. J Food Prot 65:709–725 12. PulseNet USA (2009) One-day (24-28 h) standardized laboratory protocol for molecular subtyping of Listeria monocytogenes by Pulsed Field Gel Electrophoresis (PFGE). Accesses on March 4, 2014. Available from: http://www. pulsenetinternational.org/protocols/ 13. Carpentier B, Cerf O (2011) Review— persistence of Listeria monocytogenes in food industry equipment and premises. Int J Food Microbiol 145:1–8 14. Fox EM, Leonard N, Jordan K (2011) Physiological and transcriptional characterization of persistent and nonpersistent Listeria monocytogenes isolates. Appl Environ Microbiol 77:6559–6569 15. Dalmasso M, Jordan K (2013) Process environment sampling can help to reduce the occurrence of Listeria monocytogenes in food

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processing facilities. Irish J Agric Food Res 52:93–100 Fox EM, O’Mahony T, Clancy M, Dempsey R, O’Brien M, Jordan K (2009) Listeria monocytogenes in the Irish dairy farm environment. J Food Prot 72:1450–1456 Nightingale KK, Schukken YH, Nightingale CR, Fortes ED, Ho AJ, Her Z et al (2004) Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment. Appl Environ Microbiol 70: 4458–4467 Ho AJ, Lappi VR, Wiedmann M (2007) Longitudinal monitoring of Listeria monocytogenes contamination patterns in a farmstead dairy processing facility. J Dairy Sci 90: 2517–2524 Dalmasso M, Jordan K (2014) Absence of growth of Listeria monocytogenes in naturally contaminated Cheddar cheese. J Dairy Res 81:46–53 Hunt K, Drummond N, Murphy M, Butler F, Buckley J, Jordan K (2012) A case of bovine raw milk contamination with Listeria monocytogenes. Irish Vet J 65:13 Alessandria V, Rantsiou K, Dolci P, Cocolin L (2010) Molecular methods to assess Listeria monocytogenes route of contamination in a dairy processing plant. Int J Food Microbiol 141: S156–S162 Reij MW, Den Aantrekker ED, ILSI Europe Risk Analysis in Microbiology Task Force (2004) Recontamination as a source of pathogens in processed foods. Int J Food Microbiol 91:1–11

Chapter 4 Case Study of the Use of Pulsed Field Gel Electrophoresis in the Detection of a Food-Borne Outbreak Niall De Lappe and Martin Cormican Abstract In early July 2008, a cluster of six Salmonella Agona was identified in the Republic of Ireland. A dispersed, common source outbreak was suspected. Later in July a further case was identified and the Health Protection Agency in the UK indicated that they had 32 cases of S. Agona since Feb 2008. This chapter discusses how pulsed field gel electrophoresis was used to help confirm an outbreak and to trace the source of the outbreak. Keywords PFGE, Salmonella Agona, Outbreak investigation

1

Introduction The National Salmonella, Shigella & Listeria Reference Laboratory (NSSLRL) in Ireland receives 300–500 S. enterica isolates for subtyping each year from human sources with approximately half the cases reporting a history of recent foreign travel. The majority of cases of infection are believed to arise from ingestion of contaminated foods while animal contact and person-to-person transmission play a lesser role. Subtyping supports a conclusion that many Salmonella infections in Ireland are epidemiologically unrelated although outbreaks do occur. Although more than 2,500 Salmonella serotypes exist, there are vast differences in their prevalence. Serotypes S. Enteritidis, S. Typhimurium, and monophasic Typhimurium (4,[5],12:i:-) combined account for more than half of Salmonella infections detected each year in Ireland over the last 10 years.

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_4, © Springer Science+Business Media New York 2015

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S. Agona outbreak S. Agona (4,12:f,g,s) was first isolated from cattle in Ghana in 1952 [1] and it spread worldwide in the late 1960s. Dissemination has been attributed to use of contaminated Peruvian anchovies in the production of fishmeal [2]. By 1972, it was the second most common serotype in humans in the UK [3] and the eighth in the USA [2]. S. Agona is now a common contaminant in vegetables and food animals [4] and it has caused numerous large-scale food-borne outbreaks including an outbreak associated with a flavored children’s snack in the UK in 1996 (n = 27 cases) [5]; two outbreaks in the USA due to contaminated Malt-O-Malt cereal; one in 1998 (n = 209 cases) and the other in April 2008 (n = 21 cases) [6]; infant aniseed drink preparation in Germany in 2003 (n = 42 cases) [7]; and contaminated infant formula in France in 2005 (n = 141 cases) [8]. In the first 2 weeks of July 2008, the NSSLRL noted a cluster of six S. Agona isolates. These were the first isolates of S. Agona from humans that year. S. Agona is a relatively uncommon isolate in humans in Ireland with a range of 1–6 isolates most years, apart from 2005 where there was an increase (n = 10 cases) due to an outbreak. The patients in the 2008 cluster of six isolates were from different regions of Ireland and were in the age range of 20–45. This was unusual as Salmonella infections tend to be more frequently diagnosed in the very young and very old. A dispersed, common source outbreak was suspected and a national alert was issued by the Health Protection Surveillance Centre. On July 16th, a further case was identified and an alert was issued to the UK and Northern Ireland. The Health Protection Agency in the UK indicated that they had 32 cases of S. Agona since Feb 20th. The cases were 30 in England and Wales and 2 in Northern Ireland. The isolates were designated as a new phage type PT39. The Scottish Reference laboratory (SSRL) indicated that they had detected 19 cases since May 9th (Fig. 1). An outbreak involving Ireland and the UK was declared and an international outbreak control team (OCT) was established. In Ireland PFGE had been performed on a number of S. Agona isolates from human (n = 18 isolates) and nonhuman (n = 45 isolates) sources prior to 2008. PFGE of the human isolates showed a high index of diversity with just one of the pre-2008 human isolates (S05-0707) demonstrating the SAGOXB.066 pattern, the designated name for the 2008 isolate. However, a number of food and environmental isolates, mainly from poultry sources, but also single isolates from a calf, milk filter, feed mill, and pork salami in 2007 exhibited the profile SAGOXB.066. As the strain had been demonstrated in food and food animals circulating in Ireland since at least 2003 and had not been detected in the UK prior to 2008, the source of the outbreak was suspected to originate in Ireland.

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Fig. 1 Distribution of S. Agona cases in the UK and Ireland from February 1st to September 1st, 2008, showing the dispersed nature of the outbreak

An alert was issued to the EU Early Warning Response System and the Food and Waterborne Disease Network to determine if other countries had seen a similar isolate. The NSSLRL offered to perform PFGE on isolates from other countries if requested to do so. Four S. Agona isolates from 2008 food samples, including cooked bacon, pepperoni, swine, and beef powder, had been submitted to the NSSLRL earlier in 2008. The beef powder isolate was suspected to be a result of laboratory cross-contamination in the primary laboratory as they had isolated Salmonella from their pipettes. PFGE was performed on the S. Agona food isolates using the PulseNet method [9]. Patterns from the human and food were indistinguishable (Fig. 2). The normalized images were uploaded to PulseNet Europe and matched the isolates from Scotland which had been designated SAGOXB.066 by curators in PulseNet Europe. The bacon-related S. Agona isolate was reported to have been detected in an undercooked bacon sample produced by a very major cooked meat-producing facility in Ireland in April. This company supplied ready-to-eat meat products to numerous food service outlets. A cooker failure was identified and production had

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Fig. 2 PFGE profiles of S. Agona isolates (lanes 2–4, 6–9) from both human and food sources. All had indistinguishable patterns, apart from the isolate in lane 7

been suspended. However, undercooked product had entered the chilled area for cooked meats prior to being discarded. The identification of S. Agona with the distinctive PFGE profile in this company’s product led to concern that product from that company might be associated with the outbreak. An epidemiological case control study in Ireland demonstrated that outbreak cases were 18 times more likely to have eaten food supplied from company A than controls that were not sick. Among the cases outside Ireland or the UK, a case in Finland ate steak pieces from a chain supplied by the company. S. Agona SAGOXB.066 was isolated from chicken, pork product, and beef strips obtained from outlets supplied by the company. The combination of the epidemiological and microbiological evidence provided a clear evidence base for the company to undertake temporary closure of the facility and a voluntary withdrawal of relevant products from the market. The number of new cases of infection decreased and the outbreak was declared over in October 2008. By that time a total of 163 cases had been recorded, including cases in England (n = 96 cases), Scotland (n = 34 cases), Wales (n = 11 cases), Ireland (n = 11 cases), France (n = 3 cases), Northern Ireland (n = 2 cases), Sweden (n = 2 cases), Luxembourg (n = 2 cases), Finland (n = 1 case), and Austria (n = 1 case). There were two deaths of elderly patients although it is not clear whether the Salmonella infection was the cause of death.

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The initial outbreak signal was the recognition of a cluster of six cases of an unusual serotype; however, indistinguishable PFGE patterns proved invaluable in supporting the early conclusion that this was unlikely to be a chance finding and that an outbreak was in fact in progress. Subsequent application of PFGE to food isolates provided critical support for decision makers. The decision to cease production and conduct a product recall is a difficult one. Making the decision to proceed early can prevent serious illness; however, unnecessary action can have profound economic consequences for the food-producing company, its employees, and potentially a public agency involved in making the decision. PFGE helped to make the case sufficiently persuasive to enable the agencies to advise, and the company to accept the need for prompt action. PFGE was also valuable in showing that this outbreak strain was different than the S. Agona strain responsible for a concurrent outbreak in the USA. PFGE also facilitated ruling out some temporally related S. Agona isolates which were not linked to the outbreak. Use of the standardized PulseNet protocol allowed rapid comparisons between isolates worldwide.

3

Conclusion PFGE has some limitations. PFGE profiles can change to some degree during the course of an outbreak. In this investigation, S. Agona isolates with slightly different banding patterns were detected. Interpretation of patterns can allow for appropriate interpretation of such changes. Differences in PFGE profiles may not always correlate with strain genealogies [10]. Very closely related strains may have somewhat different PFGE patterns and unrelated strains may by chance have similar patterns. In practice, and in the context of an outbreak investigation, these are manageable challenges if interpretation is made by experienced staff. Performance of PFGE is labor intensive and time consuming, so real-time PFGE of all isolates may not be feasible for reference laboratories. This could hinder outbreak investigation. In contrast to Salmonella serotyping or antimicrobial susceptibility testing, where the nomenclature is well standardized, isolates with the same PFGE pattern are often assigned different names by different groups, e.g., PulseNet USA and ECDC. Despite these disadvantages, the use of PFGE led to earlier detection and resolution of this outbreak and most likely prevented further illness in the population of Ireland and Europe more generally.

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References 1. Guinee PAM, Kampelmacher EH, Willems HMCC (1961) Six new Salmonella types, isolated in Ghana (S. volta, S. agona, S. wa, S. techimani, S. mampong and S. tafo). Antonie Van Leeuwenhoek 27:469–472 2. Clark GM, Kaufmann AF, Gangarosa EJ, Thompson MA (1973) Epidemiology of an international outbreak of Salmonella agona. Lancet 2:490–493 3. Lee JA (1974) Recent trends in human salmonellosis in England and Wales: the epidemiology of prevalent serotypes other than Salmonella typhimurium. J Hyg (Lond) 72: 185–195 4. Quiroz-Santiago C, Rodas-Suarez OR, Carlos RV, Fernandez FJ, Quinones-Ramirez EI et al (2009) Prevalence of Salmonella in vegetables from Mexico. J Food Prot 72:1279–1282 5. Threlfall EJ, Hampton MD, Ward LR, Rowe B (1996) Application of pulsed-field gel electrophoresis to an international outbreak of Salmonella agona. Emerg Infect Dis 2:130–132 6. CDC (1998) Multistate outbreak of Salmonella serotype Agona infections linked to toasted

7.

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oats cereal–United States, April-May, 1998. MMWR Morb Mortal Wkly Rep 47:462–464 Rabsch W, Prager R, Koch J, Stark K, Roggentin P et al (2005) Molecular epidemiology of Salmonella enterica serovar Agona: characterization of a diffuse outbreak caused by aniseedfennel-caraway infusion. Epidemiol Infect 133: 837–844 Brouard C, Espie E, Weill FX, Kerouanton A, Brisabois A et al (2007) Two consecutive large outbreaks of Salmonella enterica serotype Agona infections in infants linked to the consumption of powdered infant formula. Pediatr Infect Dis J 26:148–152 Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV (2001) CDC PulseNet Task Force. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis 7(3):382–389 Zhou Z, McCann A, Litrup E, Murphy R, Cormican M, Fanning S, Brown D, Guttman D, Brisse S, Achtman M (2013) Neutral genomic microevolution of a recently emerged pathogen, Salmonella enterica serovar agona. PLoS Genet 9(4):e1003471

Part II PFGE and Pathogenic Bacteria

Chapter 5 Pulsed-Field Gel Electrophoresis for Listeria monocytogenes Laura Luque-Sastre, Séamus Fanning, and Edward M. Fox Abstract Pulsed-Field Gel Electrophoresis (PFGE) is a molecular subtyping method with high discriminatory power, reproducibility, and epidemiological concordance for the subtyping of Listeria monocytogenes and other bacteria. PFGE uses rare-cutting restriction enzymes (macrorestriction) that cut the genomic DNA, usually resulting in 6–25 DNA fragments ranging between 30 and 600 kb. Bacterial cells are immobilized in agarose plugs and subsequently lysed to release genomic DNA, which is then subjected to DNA digestion. AscI and ApaI restriction enzymes are typically used for L. monocytogenes. Electrophoresis using an alternating electric field direction results in a DNA banding pattern, or fingerprint, which is used to classify isolates into different pulsotypes. PFGE is currently the CDC’s gold standard method for epidemiological studies in foodborne outbreaks. Key words Pulse-field gel electrophoresis, Discriminatory power, Reproducibility, Macrorestriction, AscI, ApaI, Fingerprint, Pulsotypes, Foodborne outbreaks

1

Introduction Listeriosis, the foodborne disease caused by Listeria monocytogenes, remains one of the main public health concerns in Europe and the United States with a mortality rate approximately 20–30 % [4]. In 1993, The Centre for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories developed PulseNet, which is a network of health and food regulatory laboratories. PulseNet has developed different standardized protocols that use PFGE for subyping a large number of foodborne pathogens for improved detection and understanding the epidemiology of outbreaks. A standardized protocol was developed for subtyping L. monocytogenes by PFGE [2, 3]. This highly discriminatory method uses two rare-cutting restriction enzymes, AscI and/or ApaI in the case of L. monocytogenes, to prepare fingerprint profiles of strains [1]. PFGE can allow the detection of genetic changes such as point

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_5, © Springer Science+Business Media New York 2015

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mutations in restriction sites, insertions, deletions, and genetic rearrangements, which result in variation in DNA banding patterns, and can be used to discriminate different strains. Although PFGE is an efficient discriminatory technique, it requires specialized equipment and is not a rapid subtyping tool [5]. However, the method allows effective discrimination of bacterial populations. Inter-laboratory comparisons with PFGE can be challenging, and to address this, standardized protocols have been created to facilitate both national and international strain comparisons [6, 7].

2

Materials Prepare all solutions using ultrapure water (prepared with Reagent Grade Type 1 Ultrapure water to attain a sensitivity of 18.2 MΩ cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at 18–25 °C (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing of waste materials.

2.1 Growth of the Cultures 2.2 Cell Suspension and Casting Plugs

1. Tryptic Soy Agar (TSA) plates or comparable nonselective media. 2. Sterile loops. 1. Sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0): Add 10 mL of 1 M Tris, pH 8.0, 2 mL of 0.5 M EDTA, pH 8.0 and dilute to 1,000 mL with sterile Ultrapure Water. 2. Sterile Cell Suspension Buffer (100 mM Tris:100 mM EDTA, pH 8.0): Add 100 mL of 1 M Tris, pH 8.0, 200 mL of 0.5 M EDTA, pH 8.0 and dilute to 1,000 mL with sterile Ultrapure water. 3. Lysozyme (20 mg/mL) stock solution. Weigh out 100 mg Lysozyme, add 5 mL TE buffer, swirl to mix and aliquot 250 μL volumes into sterile tubes, and freeze at −20 °C for future use. Once thawed, discard any unused lysozyme reagent. 4. Proteinase K (20 mg/mL) stock solution. The stock solution of Proteinase K can be prepared from the powder with sterile Ultrapure water and aliquot 250 μL volumes into sterile Eppendorf tubes and freeze at −20 °C for future use. Once thawed, discard any unused Proteinase K reagent. 5. Prepare 1 % SeaKem Gold agarose (SKG) in TE Buffer for PFGE plugs; Weigh 0.1 g (SKG) agarose into a flask, add 10.0 mL of TE Buffer; swirl gently to disperse the agarose, loosen the cap, and microwave for 30 s mix gently; and repeat for 10 s intervals until the agarose is completely dissolved. Place the flask in a water bath set to 55–60 °C and equilibrate the agarose until ready to use.

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6. Sterile polyester-fiber or cotton swabs. 7. Spectrophotometer and cuvettes. 8. 50-well disposable plug mold or 10-well reusable plug mold. 2.3 Lysis of Cells in Agarose Plugs

1. Cell Lysis Buffer (CLB; 50 mM Tris:50 mM EDTA, pH 8.0 + 1 % Sarcosyl); Add 50 mL of 1 M Tris, pH 8.0, 100 mL of 0.5 M EDTA, pH 8.0, 100 mL of 10 % Sarcosyl (NLauroylsarcosine, Sodium salt) and dilute to 1,000 mL with sterile Ultrapure water (see Note 1). 2. Proteinase K (20 mg/mL) (see Subheading 2.2, item 4).

2.4 Washing of Agarose Plugs

1. Sterile water. 2. Sterile TE Buffer (see Subheading 2.2, item 1). 3. Spatula.

2.5 Restriction Digestion of DNA in Agarose Plugs

1. CutSmart Buffer 10×. 2. AscI enzyme or ApaI enzyme. 3. Sterile blade. 4. Glass slide. 5. Spatula.

2.6 Casting of the Agarose Gel

1. Gel and Electrophoresis Running Buffer (0.5× Tris-Borate EDTA Buffer) (TBE, Table 1): Add 2.2 mL of 500 mM Thiourea and 110 mL of 10× TBE stock solution into 2,090 mL sterile Ultrapure Water. 2. 1 % SKG Agarose in 0.5× TBE: Weigh the appropriate amount of SKG into a screw cap flask: –

Mix 1.0 g agarose with 100 mL 0.5× TBE for 14 cm-wide gel form.

–

Mix 1.5 g agarose with 150 mL 0.5× TBE for 21 cm-wide gel form.

Add the appropriate amount of 0.5× TBE; swirl gently to disperse the agarose, loosen the cap, and microwave for 60 s; Table 1 Electrophoresis running buffer (0.5× TBE) Reagent

Volume (mL)

500 mM thiourea

2.2

10× TBE

110

Molecular grade water

2,090

Total volume of 0.5× TBE

2,200

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mix gently and repeat for 15 s intervals until the agarose is completely dissolved. Return the flask to 55–60 °C water bath until ready to use. 3. Gel comb. 2.7 Electrophoresis Conditions

1. PFGE equipment CHEF Mapper, CHEF-DR III, or CHEF-DR II. 2. Cooling module for PFGE system. 3. Thiourea minimum 99 %.

2.8 Staining and Documentation of the Agarose Gel

1. SYBR Safe DNA gel Stain 10× concentrate in dimethyl sulfoxide (DMSO). 2. Imaging equipment Gel Doc 1000 or equivalent. 3. BioNumerics Software.

3

Methods Carry out all procedures at 18–25 °C unless otherwise specified.

3.1 Growth of the Cultures

1. Streak an isolated colony from the test cultures onto a TSA plate (or comparable nonselective media) for confluent growth (see Note 2). 2. Incubate the plate at 37 °C for 14–18 h.

3.2 Preparation of the Plugs

1. Turn on the stationary water bath (55–60 °C) or shaker water bath or incubator at 54–55 °C. 2. Turn on the spectrophotometer at 610 nm wavelength and prepare the blank with sterile TE buffer. 3. Label 2 mL tubes with the culture numbers and transfer 2 mL of TE Buffer into each tube. Use a sterile polyester-fiber or cotton swab that has been moistened with sterile TE to remove some of the growth from the agar plate; suspend the cells in TE Buffer by spinning the swab gently so that the cells are evenly dispersed and the formation of aerosols is minimized (see Note 3). 4. Adjust the concentration of the cell suspensions to an OD610nm of 1.00 by diluting with sterile TE buffer or by adding additional cells, as required.

3.3 Casting the Plugs

1. Label the wells of the PFGE plug molds with the culture number. If reusable plug molds are used, seal the bottom of the wells with a strip of sticky tape on lower part of reusable plug mould before labeling wells. 2. Transfer 400 μL adjusted cell suspensions to labeled 1.5 mL tubes.

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3. Add 20 μL of thawed lysozyme (20 mg/mL) from the stock solution to each tube and mix by gently pipetting five to ten times. 4. Place the tubes into a 55–60 °C water bath for 10–20 min and discard any unused thawed lysozyme solution. 5. Add 20 μL of Proteinase K (20 mg/mL) from the stock solution to each tube and mix gently with a pipette tip. 6. Add 400 μL of melted 1 % SKG agarose to 400 μL cell suspension; mix by gently pipetting the mixture up and down five to ten times. Over-pipetting can cause DNA shearing. Maintain the temperature of the melted agarose by keeping the flask in a beaker of warm water (55–60 °C). 7. Immediately, dispense part of the mixture into appropriate well(s) of the reusable plug mould. Do not allow bubbles to form. Multiple plugs of each sample can be made from these amounts of cell suspension and agarose and are useful if repeat testing is required. Allow the plugs to solidify at 18–25 °C for 10–15 min. They can also be placed in the refrigerator (4 °C) for 5 min (see Note 4). 3.4 Lysis of Cells in the Agarose Plugs

1. Label 15 mL tubes with the culture numbers. 2. Calculate the total volume of CLB-Proteinase K for a final concentration of 0.1 mg/mL Proteinase K: ●

●

5 mL CLB is needed per tube. 25 μL Proteinase K stock solution (20 mg/mL) is needed per tube of the CLB.

3. Prepare the master mix by measuring the correct volume of CLB and Proteinase K into appropriate size test tube or flask and mix well. 4. Add 5 mL of CLB-Proteinase K to each labeled 15 mL tube. 5. Trim excess agarose from the top of the plugs with a scalpel, razor blade, or similar instrument. Open the reusable plug mould and transfer the plugs from the mould with a 6-mm wide spatula to the appropriately labeled tube. If disposable plug moulds are used, remove the white tape from the bottom of the mould and push out the plug(s) into the appropriately labeled tube, note that two plugs (reusable moulds) or three to four plugs (disposable moulds) of the same strain can be lysed in the same 15 mL tube. Be sure the plugs are under buffer and not on the side of the tube. 6. Remove the tape from the reusable mould. Place both sections of the plug mould, spatulas, and scalpel in 90 % ethanol, 1 % Lysol/Amphyll or other suitable disinfectant. Soak them for 15 min before washing them. Discard disposable plug moulds or disinfect them in 90 % ethanol for 30–60 min if they will be washed and reused.

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7. Place the tubes in a rack and incubate in a 54–55 °C shaker water bath or incubator for 1.5–2 h with constant and vigorous agitation (150–175 rpm). If lysing in the water bath, be sure water level is above the level of CLB in tubes. 8. Preheat enough sterile Ultrapure water to 50 °C so that the plugs can be washed twice with 10–15 mL water (200–300 mL for ten tubes). 3.5 Washing of Plugs After Cell Lysis

1. Remove the tubes from the water bath or incubator, and carefully pour off the CLB into an appropriate discard container; the plugs can be held in the tubes with a screened cap or spatula. It is important to remove all of the liquid. 2. Add 10–15 mL sterile Ultrapure water (CLRW) that has been pre-heated to 50 °C to each tube and shake the tubes in a 50 °C water bath or incubator for 10–15 min. 3. Pour off the water and repeat the wash step with preheated water (step 2) one more time. 4. Preheat enough sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0) in a 50 °C water bath so that plugs can be washed four times with 10–15 mL TE (400–600 mL for ten tubes) after beginning the last water wash. 5. Pour off the water, add 10–15 mL preheated (50 °C) sterile TE Buffer, and shake the tubes in a 50 °C water bath or incubator for 10–15 min. 6. Pour off the TE and repeat the wash step with preheated TE three more times. Decant the last wash and add 5–10 mL sterile TE. Continue with step 1 in Subheading 3.6 or store the plugs in TE Buffer at 4 °C until needed (plugs are stable for at least 4 weeks). Plugs can be transferred to smaller tubes for long-term storage. If restriction digestion is to be done the same day, complete steps 1–3 of Subheading 2.5 during the last TE wash step for optimal use of time.

3.6 Restriction Digestion of DNA in Agarose Plugs

1. Turn on the water bath to 37 °C. 2. Label 1.5 mL tubes with culture numbers. Label 3 or 4 tubes for Salmonella ser. Braenderup H9812 standards. 3. Dilute 10× CutSmart Buffer 1:10 with sterile Ultrapure (Reagent Grade Type 1) water according to Table 2. 4. Carefully remove the plug from TE with a spatula and place it in a sterile disposable Petri dish or large glass slide. 5. Cut a 2.0–2.5 mm-wide slice from the test samples with a single edge razor blade to ensure all rough edges are removed, and transfer to a tube containing diluted CutSmart Buffer (1×), ensuring the plug slice is under buffer. 6. Replace the rest of the plug in the original tube that contains 5 mL TE Buffer and store at 4 °C.

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Table 2 Prerestriction digestion 1× Reagent

μL/plug slice

μL/15 plug slices

Sterile molecular grade water

180

2,700

20

300

200

3,000

Reagent

μL/plug slice

μL/15 plug slices

Sterile molecular grade water

180

2,700

5

75

20

300

200

3,000

CutSmart buffer (10×) Total volume

Table 3 Restriction digestion with ApaI

ApaI enzyme (10 U/μL) CutSmart buffer (10×) Total volume

7. Cut three or four 2.0 mm-wide slices from the plug of the S. ser. Braenderup H9812 standard and transfer to tubes with diluted CutSmart buffer, ensuring the plugs are under the buffer. Again, replace the rest of plug in the original tube that contains 5 mL TE Buffer and store at 4 °C if not used immediately. 8. Add 200 μL diluted CutSmart Buffer (1×) to labelled 1.5 mL tubes. 9. Incubate at 18–25 °C for 15–20 min. 10. After incubation, aspirate the buffer from the plug slice using a 200–250 μL tip. Care is needed to ensure the plug slice remains intact. 11. Dilute 10× CutSmart Buffer 1:10 with Molecular Grade Water and add ApaI restriction enzyme (50 U/sample) or add AscI restriction enzyme (40 U/sample) (keep the enzymes on ice) according to Table 3 or 4. Mix in the same tube that was used for the diluted CutSmart Buffer. Keep restriction enzyme on ice at all times (see Note 5). 12. Add 200 μL of restriction enzyme mixture to each tube. Close the tubes and mix by tapping gently, ensuring the plug slices are under the enzyme mixture. 13. Incubate the samples at 25 °C overnight for ApaI or 37 °C for 3 h for AscI and XbaI (see Note 6).

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Table 4 Restriction digestion with AscI Reagent

μL/plug slice

μL/15 plug slices

Sterile molecular grade water

180

2,700

4

60

20

300

200

3,000

AscI enzyme (10 U/μL) CutSmart buffer (10×) Total volume

3.7 Casting the Agarose Gel and Loading the DNA Plugs

1. Ensure the water bath is equilibrated to 55–60 °C. 2. Turn on the cooling module (14 °C), power supply, and pump (setting of ~70 for a flow of 1 L/min). 3. Clean the PFGE equipment with preheated (55–60 °C) pure deionized water and run for a minimum of 30 min. 4. Make a volume of 0.5× Tris-Borate EDTA Buffer (TBE) that is needed for both gel and electrophoresis running buffer according to Table 1. 5. Add 2–2.2 L freshly prepared 0.5× TBE to the PFGE chamber ensuring that there are no air bubbles and that the unit is level, and then close the cover of the unit. 6. Remove restricted plug slices from the 37 °C water bath (or from the fridge). Remove any enzyme/buffer mixture and add 200 μL 0.5× TBE. Allow to stand at 18–25 °C for 5 min. 7. Remove the plug slices from the tubes and gently lay them on tissue to remove any excess buffer. Put the comb on the bench top and load the plug slices on the bottom of the comb teeth with the S. ser. Braenderup H9812 standards on teeth (lanes) 1, 5, and 10 of a 14 cm comb, or lanes 1, 8, and 15 of a 21 cm comb. Load the samples on the remaining teeth. 8. Confirm that the plug slices are correctly aligned on the bottom of the teeth. Ensure that the lower edge of the plug slice is flush against the black platform, and that there are no air bubbles. 9. Allow the plug slices to air dry on the comb for 5 min. 10. Place the gel form on a leveling table and adjust until perfectly leveled before pouring the gel. Position the comb holder so that the front part (side with small metal screws) and teeth face the bottom of the gel and the bottom edge of the comb is 2 mm above the surface of the gel platform and then carefully pour the agarose (cooled to 55–60 °C) into the gel mould. 11. Remove the comb after the gel solidifies (this will take approximately 20 min).

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12. Unscrew and remove the end gates from the gel mould; remove excess agarose from the sides and bottom of the casting platform with a tissue. 13. Keep the gel on the casting platform and carefully place it inside the black gel frame in the electrophoresis chamber. Close the cover of the chamber. 3.8 Electrophoresis Conditions

1. Set up the corresponding conditions for your electrophoresis equipment: ●

Conditions for CHEF Mapper: ●

Auto Algorithm.

●

49 kb: low MW.

●

450 kb: high MW.

●

●

●

●

●

Select default values except where noted by pressing “enter.” Set run time to 20–21 h (see Note 7). Default values: Initial switch time = 4.0 s; Final switch time = 40.0 s.

Conditions for CHEF-DR III: ●

Initial switch time: 4.0 s.

●

Final switch time: 40.0 s.

●

Voltage: 6 V.

●

Included angle: 120°.

●

Run time: 18–19 h (see Note 7).

Conditions for CHEF-DR II: ●

Initial A time: 4.0 s.

●

Final A time: 40.0 s.

●

Start ratio: 1.0 (if applicable).

●

Voltage: 200 V.

●

Run time: 19–20 h (see Note 7).

2. Make a note of the initial milliamp (mA) reading on the instrument. The initial mA should be between 110 and 150 mA. A reading outside of this range may indicate that the 0.5× TBE buffer was prepared improperly and the buffer should be remade. 3.9 Staining and Documentation of the PFGE Agarose Gel

1. When electrophoresis run is over, turn off the equipment and remove the gel. 2. Dilute 40 μL of SYBR® Safe (see Note 8) with 400 mL of molecular grade water and stain gel with this solution for 30 min in a covered container.

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3. Destain the gel in approximately 400 mL of Ultra pure water for 60–90 min, changing the water every 20 min. 4. Capture the image on Gel Doc 1000 or equivalent documentation system. If the background interferes with the resolution, destain for an additional 30–60 min. 5. Follow the directions given with the imaging equipment to save the gel image as an *.img or *.1sc file. Convert this file to *.tif file for analysis with the BioNumerics software program. 6. Drain the buffer from the electrophoresis chamber and discard it. Rinse the chamber with 2 L of pre-heated (50–55 °C) molecular grade water and flush the lines with water by letting the pump run for 15–20 min before draining the water from chamber and hoses.

4

Notes 1. To dissolve the Sarcosyl easily, add 10 g of Sarcosyl directly to the Cell Lysis buffer when preparing the reagent. This replaces adding 100 mL of a 10 % Sarcosyl solution, as Sarcosyl solution can be difficult to dissolve into solution when prepared at 10 %. 2. It is recommended that a storage vial of each culture be created. This will ensure that the same colony can be retested if necessary. 3. Keep cell suspensions on ice if you have more than six cultures to process or refrigerate cell suspensions if you cannot adjust their concentration immediately. 4. Four plugs can be made from these amounts of cell suspension and agarose. The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as possible in order to minimize premature cell lysis. If large numbers of samples are being prepared, it is recommended that they be processed in batches of around ten samples at a time. Once the first batch of isolates are in the cell lysis incubation, then start preparing the cell suspensions for the next group of samples, and so on. All batches can be lysed and washed together, since additional lysis time will not affect the initial batches 5. ApaI is recommended as the secondary enzyme for the analysis of L. monocytogenes isolates. The use of a secondary enzyme is useful in situations where the PFGE patterns obtained with the primary enzyme are also indistinguishable (e.g., for the identification of related clonal groups). 6. Check the conditions for the restriction enzymes as different suppliers may specify different temperatures.

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7. The electrophoresis running times recommended above are based on the equipment and reagents used at the CDC. Run times may be different in your laboratory and will have to be optimized for your gels, so that the lowest band in the S. ser. Braenderup H9812 standard migrates within 1.0–1.5 cm of the bottom of the gel. 8. Other staining solutions can be used, such as ethidium bromide or GelRed. References 1. Wiedmann M (2002) Molecular subtyping methods for Listeria monocytogenes. J AOAC Int 85:524–531 2. Graves LM, Hunter SB, Ong AR, SchoonmakerBopp D, Hise K, Kornstein L, Dewitt WE, Hayes PS, Dunne E, Mead P, Swaminathan B (2005) Microbiological aspects of the investigation that traced the 1998 outbreak of listeriosis in the United States to contaminated hot dogs and establishment of molecular subtypingbased surveillance for Listeria monocytogenes in the PulseNet network. J Clin Microbiol 43: 2350–2355 3. Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV, Force CPT (2001) PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis 7:382–389 4. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M-A, Roy SL, Jones JL, Griffin PM (2011) Foodborne illness acquired in the

United States–major pathogens. Emerg Infect Dis 17:7–15 5. Murphy M, Corcoran D, Buckley JF, O’Mahony M, Whyte P, Fanning S (2007) Development and application of multiple-locus variable number of tandem repeat analysis (MLVA) to subtype a collection of Listeria monocytogenes. Int J Food Microbiol 115: 187–194 6. PulseNet USA (2009) One-day (24–48 h) standardized laboratory protocol for molecular subtyping of Listeria monocytogenes by Pulsed-Field Gel Electrophoresis (PFGE). http://www. pulsenetinternational.org/assets/PulseNet/ uploads/pfge/PNL04_ListeriaPFGEProtocol. pdf 7. Fox EM, DeLappe N, Garvey P, Cormican M, Leonard N, Jordan K (2012) PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland. J Med Microbiol 61:540–547

Chapter 6 Pulsed-Field Gel Electrophoresis (PFGE) for Pathogenic Cronobacter Species Qiongqiong Yan and Séamus Fanning Abstract Pulsed-field gel electrophoresis (PFGE) is a molecular-based subtyping strategy that uses a suitable DNA restriction endonuclease enzyme to cut genomic DNA into several large linear fragments, that can be separated based on their sizes. PFGE has been successfully applied to the subtyping of many pathogenic bacteria, including Cronobacter species, and it is commonly considered as a “gold standard” in epidemiological studies. Key words Cronobacter, Pulsed-field gel electrophoresis, Subtyping, Epidemiology, Genetic fingerprint

1

Introduction Cronobacter species are opportunistic pathogens that can cause necrotizing enterocolitis, bacteraemia, and meningitis, predominantly in neonates. The bacterium has been isolated from a range of food sources including dairy-based foods, dried meats, water, rice, and others [1–3]. Controlling the microbiological load and understanding the epidemiology would contribute positively towards a reduction in the health risk to vulnerable individuals. Molecular approaches have always been regarded as useful tools to extend our understanding of the epidemiology of an organism. These methods often require the analysis of a DNA fragmentation pattern or fingerprint produced following the enzymatic digestion of template DNA (including the chromosome and any plasmids) purified from a bacterium of interest. The latter requires the resolution of the DNA fingerprint using a gel-based protocol. Conventional agarose gel electrophoresis is incapable of separating DNA molecules of greater than 20 kbp. It was not until the development of pulsed-field gel electrophoresis (PFGE) by Schwartz and Cantor in 1984 [4] that the application of the method gained widespread use. This analytical approach introduced perpendicularly

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_6, © Springer Science+Business Media New York 2015

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oriented, non-uniform, alternately pulsed-electrical fields, capable of separating very large molecular weight DNA molecules with greater resolution. PFGE has been commonly used as a “gold standard” in epidemiological studies to answer the question of whether isolate A is related to isolate B by comparing their respective PFGE fingerprint profiles. Mullane et al. [5] applied PFGE subtyping for Cronobacter species as a component part of a monitoring program to locate points of contamination, investigate clonal persistence, and identify possible dissemination routes along the powdered infant formula processing chain in 2007. Similar applications of this approach followed and more recently culminated in the development of a standardized approach to PFGE of Cronobacter species that is now included as a validated PulseNet protocol [6–22]. Figure 1 shows a flow chart of the main steps in the PFGE protocol. Bacterial genomic DNA is recovered and lysed in agarose plugs, following multiple washing steps using sterile water, and Tris-Ethylenediaminetetraacetic acid (TE) buffer. Total genomic DNA is then digested with a suitable restriction endonuclease enzyme and the large linear DNA fragments are separated based on their size using a pulsed electric field. Finally, the gel is stained and

Fig. 1 A flow chart describing the PFGE protocol of pathogenic Cronobacter species Stage 1, preparing cell suspensions; Stage 2, preparing PFGE plugs; Stage 3, Lysis and washing of PFGE plugs; Stage 4, restriction digestion of DNA; Stage 5, gel electrophoresis of restricted DNA; Stage 6, PFGE gel visualization and analysis

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the DNA fingerprint photographed before digitizing it into a tiff image for uploading to BioNumerics for subsequent analysis. If two isolates were clonal, the DNA fingerprint profiles would be indistinguishable; otherwise, DNA banding patterns will vary by between a few and many bands. PFGE is highly reproducible and the patterns produced are easier to interpret compared to other techniques. Theoretically, all bacteria are typeable. The PFGE protocol for Cronobacter species is described in detail in this chapter.

2

Materials All solutions are prepared using 18 MΩ ultrapure deionized water and analytical grade reagents. All reagents are stored at 18–22 °C (unless otherwise indicated). Diligently follow all waste disposal regulations when disposing waste materials.

2.1 Making PFGE Plugs Components

1. Prepare 1 M Tris–HCl, pH 8.0: Add 100 mL water to a 1-L graduated cylinder or glass beaker. Weight 121.1 g Tris base and transfer to the graduated cylinder or glass beaker. Add water to a volume of 900 mL. Dissolve and adjust the pH to 8.0 by adding concentrated HCl (see Note 1). Make up to a final volume of 1 L with water and sterilize by autoclaving. 2. Prepare 0.5 M EDTA, pH 8.0: Add 100 mL water to a 1-L graduated cylinder or glass beaker. Weight 186.1 g Ethylenediaminetetraacetic acid (EDTA) and transfer to the graduated cylinder or glass beaker. Add water to a volume of 900 mL. Dissolve and adjust to pH 8.0 with NaOH (see Note 2). Make up to a final volume of 1 L with water and sterilize by autoclaving. 3. TE Buffer: Add 10 mL of prepared 1 M Tris–HCl (pH 8.0) and 2 mL prepared 0.5 M EDTA (pH 8.0) into 988 mL water to generate 1 L TB buffer. Sterilize by autoclaving. This yields a concentration of 10 mM Tris and 1 mM EDTA, at pH 8.0. 4. Prepare casting agarose: Weight 0.125 g Pulsed-Field Certified Agarose (see Note 3) into a 100 mL screw top flask. Add 11.75 mL TE buffer into the same flask. Swirl gently to disperse the agarose. Heat the flask using a Microwave oven until the agarose is completely dissolved. Place the flask into a 55–60 °C water bath. 5. Prepare 20 % [w/v] Sodium dodecyl sulfate (SDS): Weight 0.2 g SDS and transfer to 0.8 mL water (see Note 4). 6. Cell Suspension Buffer (CSB): Add 3 mL of prepared 1 M Tris–HCl (pH 8.0) and 6 mL of the prepared 0.5 M EDTA (pH 8.0) into 21 mL water to generate 30 mL CSB. Sterilize by autoclaving. This yields a concentration of 100 mM Tris and 100 mM EDTA, at pH 8.0.

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7. PFGE plug mold. 8. Spectrophotometer. 9. Two water baths: a stationary water bath at 55 °C and a shaking water bath at 54 °C. 10. Proteinase K, 20 mg/mL, freshly prepared each time. 11. SDS 20 % [w/v], freshly prepared each time. 12. 1 % [w/v] Pulsed-Field Certified Agarose, prepared as required. 2.2 DNA Lysis and Digestion Components

1. Cell lysis buffer (CLB): Add 6.25 mL of sterilized 1 M Tris– HCl (pH 8.0), 12.5 mL sterilized 0.5 M EDTA (pH 8.0), and 12.5 mL 10 % [w/v] Sarcosyl into 93.75 mL water to generate 125 mL CLB. This yields a concentration of 50 mM Tris, 50 mM EDTA, pH 8.0, 1 % [w/v] Sarcosyl (see Note 5), 0.1 mg/mL proteinase K. 2. Proteinase K/CLB master mix: Each sample requires 5 mL of CLB and 25 μL Proteinase K, which yields a final concentration of 0.1 mg/mL Proteinase K. 3. DNA restriction digestion mix: 1× NE Buffer 4 (available commercially), 50 U XbaI, 1× bovine serum albumin (BSA), and water. Total volume of the reaction is 200 μL. 4. Restriction enzyme: XbaI is a primary restriction enzyme, and SpeI is a secondary restriction enzyme.

2.3 Running Gel and Visualizing Components

1. Prepare 10× Tris-Borate EDTA buffer (TBE): Add 100 mL water to a 1-L graduated cylinder or glass beaker. Weigh 108 g Tris base, as well as 55 g boric acid and transfer to the graduated cylinder or glass beaker. Add 40 mL 0.5 M EDTA (pH 8.0). Make up to a final volume with 1 L water and sterilize by autoclaving. 2. Prepare 3 M Thiourea solution: Add 50 mL water to a 100 mL graduated cylinder or glass beaker. Add 22.83 g of Thiourea to the container and add water to a volume of 100 mL. Mix well. 3. Running buffer: Add 150 mL 10× TBE buffer to 2,850 mL water, supplemented with 50 μL 3 M Thiourea solution. This yields 3 L of running buffer with 0.5× TBE buffer and 50 μM Thiourea. Alternatively, 2 L of running buffer can be applied. Prepare fresh and discard after use. 4. Casting agarose gel: Weight 1.5 g Pulsed-Field Certified Agarose and transfer to a 500 mL screw top flask. Add 150 mL 0.5× TBE to the same flask. Swirl gently to disperse the agarose. Heat the flask using a microwave oven until the agarose is completely dissolved (see Note 6). Place the flask into a 55–60 °C water bath until needed. 5. Standard casting stand and 15-well comb.

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6. The PFGE electrophoresis conditions: Select the following conditions for the CHEF Mapper XA PFGE system. Auto Algorithm, with low MW of 30-kbp, high MW of 70 kbp; Initial switch time: 2.16 s, Final switch time: 63.8 s; Voltage: 6 V; Included angle: 120°; Run time 20 h. 7. Staining buffer: Add 40 μL of SYBRSafe DNA Gel Stain (see Note 7) to 400 mL water in a tray or plastic box. Cover the container with kitchen foil to protect it from light. 8. CHEF Mapper XA Pulsed-Field Electrophoresis System. CHEF DR-II or CHEF DR-III system is the alternative PFGE system. 9. Gel Logic 1500 system or any other equivalent systems for visualization of resulting agarose gels. 10. Personal computer with BioNumerics software.

3

Methods Cronobacter species is an opportunistic pathogen that can cause serious disease in individuals with a weak-immune system. Always use Biosafety Level 2 practices (at a minimum) and extreme caution when transferring and handling bacterial strains. Work in a biological safety cabinet when processing a large number of samples. Disinfect and carefully dispose of all plastic ware and glassware that come into contact with the bacterial cultures in a safe manner. Carry out all procedures at 18–22 °C unless otherwise specified.

3.1 Preparing Cell Suspension

1. Streak an isolated bacterial colony onto a previously prepared Trypticase Soy Agar (TSA) plate, and incubate at 37 °C overnight (see Note 8). Streak Salmonella Braenderup H9812 onto a TSA plate and use as a molecular weight standard during PFGE analysis. 2. Switch on a shaking water bath (54 °C) and a stationary water bath (55–60 °C). 3. Place the Pulsed-Field Certified Agarose and SDS into the 55–60 °C water bath until required. 4. Disinfect the reusable plug mold for 30–60 min using 90 % [v/v] ethanol (see Note 9). 5. Transfer 1.5 mL of CSB into labeled 1.5 mL microcentrifuge tubes. 6. Use a sterile cotton swab to pick individual colonies from the freshly cultured TSA plate and suspend them in CSB (see Note 10). 7. Transfer the cell suspension into a cuvette and read the Optical Density (OD) at a wavelength of 610 nm in a spectrophotom-

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eter. Adjust the OD610nm to 1.4 (see Note 11) and transfer the suspension to a fresh sterile 1.5 mL microcentrifuge tube. 3.2 Preparing PFGE Plugs

1. Dry the plug mold and assemble it. Label the plug mold as well as new sterile 1.5 mL microcentrifuge tubes with sample numbers. 2. Add 20 μL of Proteinase K (see Note 12) to each labeled tube. 3. Add 625 μL of the SDS to the 1 % [w/v] Pulsed-Field Certified Agarose. 4. Transfer 400 μL of the SDS/Agarose mix into the tube with Proteinase K solution. Transfer 400 μL of the corresponding cell suspensions into the SDS/Agarose/PK mix. Mix by pipetting up and down three times (see Note 13). Immediately dispense the cell–agarose mixture into the appropriate well of the plug mold and allow the plugs to solidify at 18–22 °C for 10–15 min (see Note 14).

3.3 Lysis of PFGE Plugs

1. Add 5 mL of Proteinase K/CLB master mix to labeled 15 mL tubes. 2. Carefully transfer the agarose plugs from the mold to the appropriate labeled tubes (see Note 15). 3. Place the tubes in a 54 °C shaking water bath for 90 min with constant, vigorous shaking at 175–200 rpm (see Note 16). 4. Preheat sterile purified water and TE Buffer to 50 °C for the washing of the agarose plugs after cell lysis.

3.4 Washing of PFGE Agarose Plugs

1. Remove the tubes from the water bath and reset the shaking water bath to 50 °C when the lysis finishes (see Note 17). 2. Carefully pour off the lysis buffer into an appropriate discard container. The plugs can be captured in tubes with a screened cap (see Note 18). 3. Add 10 mL of the preheated sterile water to the tubes. Place the tubes back in the 50 °C shaking water bath for 10 min. 4. Pour off the water from the plugs and repeat steps 2 and 3 one more time. 5. Pour off the remaining water and add 10 mL sterile TE buffer. Place the tubes back into the 50 °C shaking water bath for 15 min. 6. Pour off TE buffer and repeat step 5 two more times. 7. Discard TE buffer and add 5 mL sterile TE to the tubes (see Note 19). 8. Continue with “Restriction Digestion” or store the plugs at 4 °C until needed. The lysed PFGE plugs can be kept for up to 6 months.

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1. Turn on a heating block and set temperature to 37 °C. 2. Label 1.5 mL microcentrifuge tubes with sample names. 3. Cut the plugs into 5 × 3 mm slices using a sharp knife (see Note 20) and place into the respective tubes (include four samples for S. Braenderup as the molecular weight biomarker). Replace the remainder of the plug into the 5 mL TE buffer and store at 4 °C. 4. Prepare the restriction enzyme master mix. 5. Add 200 μL restriction digestion master mix into each tube. Submerge the plug slice into the enzyme mix (see Note 21). 6. Incubate the sample and biomarker plug slices in a 37 °C heating block for 3 h (see Note 22). 7. If the plug slices are to be loaded into the wells on the same day, continue with steps 1 and 2 in Subheading 3.6, approximately 1 h before the restriction digest reaction finishes. Alternatively, the plugs can be stored in 0.5× TBE buffer at 4 °C and run on a PFGE gel within 24 h.

3.6 Casting the Agarose Gel

1. Prepare 150 mL casting agarose gel and leave the flask in the 55–60 °C water bath. 2. Assemble and balance the gel casting platform (see Note 23). 3. After enzyme digestion has finished, remove the restriction enzyme mixture and add 200 μL 0.5× TBE to each tube for 10 min to stop the reaction. 4. Remove the plug slice from the TBE excess buffer and place it on the bottom of petri dish. Use a tissue to soak up the buffer without touching the plug slice (see Note 24). 5. Place the S. Braenderup onto lanes 1, 6, 11, and 15 of the comb. Spread the sample slices on the rest of the comb (for a 15 lane comb). 6. Sit the comb on the gel casting platform (see Note 25). 7. Carefully pour the molten agarose into the gel mold from a corner and let it run around the comb itself. Allow the gel to set for about 30–45 min (see Note 26).

3.7 Gel Electrophoresis of Restricted DNA

1. Add the remaining 0.5× TBE Buffer into the CHEF Mapper XA system. 2. Turn on the pump and ensure there are no air bubbles. Turn on the cooling system and set the temperature to 14 °C. 3. Carefully remove the comb and the white edges of the mold. Place the black platform with the gel into the electrophoresis chamber when the running buffer is at 14 °C. 4. Program the instrument and run the electrophoresis for the established voltages, times and pulses, with a consistent electrophoresis buffer temperature of 14 °C (see Note 27).

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3.8 PFGE Gel Visualization and Analysis of Fingerprint Patterns

1. Prepare the staining buffer using SYBRSafe DNA gel stain buffer (see Note 28). 2. Turn off the cooling system when the run has been completed. 3. Place the gel in the staining buffer for 30 min (see Note 29). 4. Destain the gel for 30 min using water (see Note 30). 5. The gel image is visualized and captured under UV light (see Note 31). 6. A Tiff image can be saved and uploaded to BioNumerics for analysis, where a dendrogram is then created using the DICE coefficient and the unweighted pair group method with arithmetic mean (UPGMA) algorithm. 7. Empty the running buffer from the CHEF Mapper XA system and wash the tank using prewarmed 55 °C water for 20 min. Drain the water afterwards for instrument maintenance.

4

Notes 1. Tris can be dissolved faster provided the water is warmed to about 37 °C. Concentrated HCl (12 M) can be used to narrow the gap from the starting pH to the required pH (8.0). A total volume of 42 mL concentrated HCl can be added to make up the final pH of 8.0. From then on, it would be better to use a series of HCl solutions (e.g., 6 M or 1 M) with lower ionic strengths to avoid a sudden drop in pH below the required pH. Critical step: Allow the solution to cool to 18–22 °C before making final adjustments to the pH. The pH of Tris solutions is temperature-dependent and decreases approximately 0.03 pH units for each 1 °C increase in temperature. 2. Approximately 20 g NaOH pellets need to be added into the solution. Critical step: The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH. 3. SeaKem® Gold Agarose is recommended in the PulseNet protocol. However, alternate types of agarose for casting gels and making plugs suggested by PulseNet include Agarose III, Certified Megabase Agarose, as well as PFGE Agarose. 4. Place the 20 % [w/v] SDS solution in 55–60 °C water bath to help with the dissolving. 5. Add 10 g N-Lauroylsarcosine sodium salt to 90 mL water to make up 10 % [w/v] Sarkosyl solution. A 55–60 °C water bath or autoclaving will help with the dissolving. 6. Make sure the agarose is fully dissolved. Gently shake the flask to observe the movement of the liquid, which will make it easier

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Fig. 2 Incomplete dissolve of agarose gel resulting in white dots on the resulting gel image (marked in yellow circle). Lane S, Salmonella Braenderup H9812; Lane 2–5, samples 1–4; Lane 7–10, samples 5–8; Lane 12–14, samples 9–11. The arrow link represents a cluster of identical isolates

to observe if there are particles remaining in the solution. Otherwise, the particles will be poured into the agarose gel and present on the resulting gel as white dots (Fig. 2). The time and temperature required to dissolve the agarose are dependent on the size of the container and the specifications of the microwave. 7. The same amount of ethidium bromide is used in the PulseNet protocol. However, ethidium bromide is toxic and a mutagen. SYBRSafe DNA Gel Stain and GelRed are the less hazardous alternatives suggested by PulseNet. 8. Fresh overnight plates are recommended because undue stress to bacterial cells prior to casting the plug can cause premature cell lysis resulting in DNA degradation (smearing). Do not grow cultures on selective media. 9. The reusable plug mold needs to be disinfected/sterilized for 30–60 min using 90 % [v/v] ethanol before use or alternatively use disposable plug molds.

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10. Suspend the cells in CSB by spinning the swab gently against the wall of the microcentrifuge tube. Make sure that the cells are well suspended in CSB before measuring the optical cell density (OD) in a spectrophotometer. Squeeze the cell suspension from the cotton swab before throwing it away. Otherwise you could end up losing half of the volume. Alternatively, use 2 mL CSB to start with. The minimum volume of the cell suspension needed will depend on the minimum test volume of the cuvettes; however, the minimum volume for next step of the experiment is 400 μL. 11. The cell suspensions can be adjusted either by adding additional cells or diluting with sterile CSB. Do not centrifuge or vortex the cell suspensions. The PulseNet protocol indicates an absorbance (Optical Density) of 1.00 (range of 0.8–1.0); however, it is recommended that each laboratory may need to establish its optimal concentration for satisfactory results. Keep the suspensions on ice if you have more than six samples to process or refrigerate the cell suspensions if you are unable to adjust the concentration immediately. If you observe dark bands in the wells or thick blurry bands in lanes, the cell concentration was too high (Fig. 3).

Fig. 3 Cell concentration too high. Dark bands and thick blurred bands are evident in these lanes. Lane S, Salmonella Braenderup H9812; Lane 2–5, samples 12–15; Lane 7–10, samples 16–19; Lane 12–14, samples 20–22. Each arrow link represents a cluster of identical isolates

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12. Proteinase K solutions of 20 mg/mL are available commercially. Or you can use sterile water to dissolve the powder of Proteinase K and make up 20 mg/mL stock solution. It is recommended to divide them into 300–500 μL aliquots and store in a freezer (−20 °C) until use. Keep Proteinase K solutions on ice at all times for the best results. 13. Keep the SDS/Agarose mix in a molten state by placing the flask in a 55–60 °C water bath or a beaker of boiled water. Do not over-pipette, as this may cause DNA shearing. 14. Load the plugs slowly to avoid air bubbles. Leaning the pipette tips on the inside wall could help with the smooth loading. Do not move the plug mold until the plugs become solid. The status of the plugs can be checked from the tube, where cell suspensions are premixed with SDS/Agarose/Proteinase K. Or alternatively, leave the plug mold in the refrigerator (4 °C) for 5 min. To minimize premature cell lysis, the preparation of the cell suspensions and casting plugs needs to be performed as quickly as possible. If large numbers of samples are being prepared, it is recommended to process each batch of ten samples. Once the first batch is in the lysis incubation, then move to the next batch for cell suspension preparation. All batches can be lysed and washed at the same time in the end because the extension of lysis time will not affect the initial batch. The agarose remaining can be maintained at 18–22 °C and reused not more than twice because repeated heating results in loss of fluid and an increase of the agarose concentration. 15. Invert a couple of times to soak the plug(s). Make sure that each plug is totally submerged in the lysis buffer and that they are not on the side of the tube. Two plugs of the same strain can be lysed in the same 15 mL tube. Discard or disinfect the extra agarose plug mold appropriately. Soak the reusable plug mold in 90 % [v/v] ethanol for 15 min before washing them. Disinfect them in 90 % [v/v] ethanol for 30–60 min if they will be washed and reused. 16. Ensure the level of water in the water bath is above the level of lysis buffer in the tube. Incomplete lysis will result in incomplete restriction, smearing, and significant fluorescence in the plug slice. 17. Most researchers carry out the washing at 54 °C and find the plugs to be sufficiently stable. However, if the plugs are nicked along the edges or breaking, you have to lower the water bath temperature to 50 °C for the washing step. 18. Mind the plug carefully during each wash step. Check the side of the tube each time you empty the washing buffer as the plug

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can easily be overlooked and may get washed away. It is important to remove the washing buffer completely. 19. Inadequate washing typically results in incomplete restriction or smearing. If you suspect plugs were not washed enough, wash plugs two more times with TE buffer. Restrict and run another plug slice. 20. Use a marked Petri dish to cut the plug slices. Plugs that are cut too small can easily be damaged and result in band distortions. Plugs that are cut too large can result in thick indistinct bands that are difficult to analyze. 21. Be careful not to damage the plug slice with the pipette tip. Keep the restriction enzyme on ice at all times. It is recommended that the 1× BSA be added to all enzyme restriction mixtures in order to minimize the incidence of incomplete restriction. 22. Incomplete restriction will result in “shadow” or “ghost” bands on the gel (Fig. 4, labeled in yellow box). This could be caused by expired enzyme and/or buffer, poor plug quality (Proteinase

Fig. 4 Shadow or ghost bands caused by incomplete restriction (marked in yellow box). Lane S, Salmonella Braenderup H9812; Lane 2–5, samples 23–26; Lane 7–10, samples 27–30; Each arrow link represents a cluster of identical isolates

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K not removed from plug), DNA concentration too high, enzyme concentration too low, or incorrect incubation temperature of the buffer. 23. The gel casting platform needs to be assembled and sealed properly. Any error will result in the gel leaking, which will then lead to incomplete coverage of the plug slice in the gel. 24. The plug slices need to be dried properly. If the slices are too wet, they could easily be washed away when casting the gel. But do not over dry. Allow the plug slices to air dry for 5–10 min. 25. Be sure the plug slice is not curved or at an angle. 26. Pour the gel slowly to allow the agarose to move around without washing away the gel slices on the comb. Do not disturb the gel after it is poured and especially while still molten. Remove air bubbles using clean pipette tips immediately after the gel is poured and before it solidifies. 27. The program is particular to the instrument. Adapt and optimize the program from the PulseNet Salmonella or E. coli protocol if you are using the CHEF DR-II or CHEF DR-III PFGE system. You will have to optimize your gels so that the lowest band in the S. Braenderup H9812 migrates 1.0–1.5 cm from the bottom of the gel. If the run time is too short, it might cause a compressed pattern, decreased resolution of closely migrating bands, or compromised normalization. If the run time is too long, the bottom band of the standard will run off the gel, and you won’t be able to perform normalization. 28. Cover the staining buffer container with kitchen foil and leave it in the dark, as the stain solution is light sensitive. The staining buffer can be reused for six to eight times before discarding. 29. Keep the container in the dark during staining. 30. If staining with GelRed or SYBRSafe DNA Gel Stain, you can destain a couple of times more or longer time if you wish. It helps to get a clear background. 31. Compare the Salmonella Braenderup H9812 with the standard pattern from PulseNet and make sure that all the bands are separated appropriately (Fig. 5). If they are, carry on with the analysis in BioNumerics. If not, the PFGE assay was not working. Go backward step-by-step to work out the possible reasons.

Acknowledgements This work is supported by the University College Dublin (UCD) and China Scholar Council (CSC).

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Fig. 5 A PFGE fingerprinting gel image. Lane S, Salmonella Braenderup H9812; Lane 2–5, samples 31–34; Lane 7–10, samples 35–38; Lane 12–14, samples 39–41. The arrow link represents a cluster of identical isolates References 1. Baumgartner A, Grand M, Liniger M, Iversen C (2009) Detection and frequency of Cronobacter spp. (Enterobacter sakazakii) in different categories of ready-to-eat foods other than infant formula. Int J Food Microbiol 136(2): 189–192 2. Chap J, Jackson P, Siqueira R, Gaspar N, Quintas C, Park J, Osaili T, Shaker R, Jaradat Z, Hartantyo SH, Abdullah Sani N, Estuningsih S, Forsythe SJ (2009) International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods. Int J Food Microbiol 136(2):185–188 3. Healy B, Cooney S, O’Brien S, Iversen C, Whyte P, Nally J, Callanan JJ, Fanning S (2010) Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen. Foodborne Pathog Dis 7(4):339–350 4. Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37(1):67–75 5. Mullane NR, Whyte P, Wall PG, Quinn T, Fanning S (2007) Application of pulsed-field gel electrophoresis to characterise and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility. Int J Food Microbiol 116(1):73–81

6. El-Sharoud WM, El-Din MZ, Ziada DM, Ahmed SF, Klena JD (2008) Surveillance and genotyping of Enterobacter sakazakii suggest its potential transmission from milk powder into imitation recombined soft cheese. J Appl Microbiol 105(2):559–566 7. Kima K, Janga SS, Kima SK, Parkb JH, Heuc S, Ryu S (2008) Prevalence and genetic diversity of Enterobacter sakazakii in ingredients of infant foods. Int J Food Microbiol 122(1–2): 196–203 8. Mullane N, Healy B, Meade J, Whyte P, Wall PG, Fanning S (2008) Dissemination of Cronobacter spp. (Enterobacter sakazakii) in a powdered milk protein manufacturing facility. Appl Environ Microbiol 74(19):5913–5917 9. Pei X, Guo Y, Liu X (2008) Study on the molecular typing of Enterobacter sakazakii with pulsed-field gel electrophoreses. Wei Sheng Yan Jiu 37(2):179–182, 186 10. Proudy I, Bougle D, Coton E, Coton M, Leclercq R, Vergnaud M (2008) Genotypic characterization of Enterobacter sakazakii isolates by PFGE, BOX-PCR and sequencing of the fliC gene. J Appl Microbiol 104(1):26–34 11. Townsend SM, Hurrell E, Caubilla-Barron J, LocCarrillo C, Forsythe SJ (2008) Characterization

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13.

14.

15.

16.

17.

of an extended-spectrum beta-lactamase Enterobacter hormaechei nosocomial outbreak, and other Enterobacter hormaechei misidentified as Cronobacter (Enterobacter) sakazakii. Microbiology 154(Pt 12):3659–3667 Anonymous (2009) Cronobacter species isolation in two infants—New Mexico, 2008. Morb Mortal Wkly Rep 58(42):1179–1183 El-Sharoud WM, O'Brien S, Negredo C, Iversen C, Fanning S, Healy B (2009) Characterization of Cronobacter recovered from dried milk and related products. BMC Microbiol 9:24 Hein I, Gadzov B, Schoder D, Foissy H, Malorny B, Wagner M (2009) Temporal and spatial distribution of Cronobacter isolates in a milk powder processing plant determined by pulsed-field gel electrophoresis. Foodborne Pathog Dis 6(2):225–233 Molloy C, Cagney C, O'Brien S, Iversen C, Fanning S, Duffy G (2009) Surveillance and characterisation by pulsed-field gel electrophoresis of Cronobacter spp. in farming and domestic environments, food production animals and retail foods. Int J Food Microbiol 136(2): 198–203 Terragnoa R, Salvea A, Pichela M, Epszteynb S, Brengia S, Binszteina N (2009) Characterization and subtyping of Cronobacter spp. from imported powdered infant formulae in Argentina. Int J Food Microbiol 136(2): 193–197 Craven HM, McAuley CM, Duffy LL, Fegan N (2010) Distribution, prevalence and persistence

18.

19.

20.

21.

22.

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of Cronobacter (Enterobacter sakazakii) in the nonprocessing and processing environments of five milk powder factories. J Appl Microbiol 109(3):1044–1052 Miled-Bennour R, Ells TC, Pagotto FJ, Farber JM, Kerouanton A, Meheut T, Colin P, Joosten H, Leclercq A, Besse NG (2010) Genotypic and phenotypic characterisation of a collection of Cronobacter (Enterobacter sakazakii) isolates. Int J Food Microbiol 139(1–2): 116–125 Simón M, Sabaté S, Cristina Osanz A, Bartolomé R, Dolores Ferrer M (2010) Investigation of a neonatal case of Enterobacter sakazakii infection associated with the use of powdered infant formula. Enferm Infecc Microbiol Clin 28(10):713–715 Mshana SE, Gerwing L, Minde M, Hain T, Domann E, Lyamuya E, Chakraborty T, Imirzalioglu C (2011) Outbreak of a novel Enterobacter sp. carrying blaCTX-M-15 in a neonatal unit of a tertiary care hospital in Tanzania. Int J Antimicrob Agents 38(3): 265–269 Ivy RA, Farber JM, Pagotto F, Wiedmann M (2013) International Life Science Institute North America Cronobacter (formerly Enterobacter sakazakii) isolate set. J Food Prot 76(1): 40–51 Müller A, Stephan R, Fricker-Feer C, Lehner A (2013) Genetic diversity of Cronobacter sakazakii isolates collected from a Swiss infant formula production facility. J Food Prot 76(5): 883–887

Chapter 7 Pulsed-Field Gel Electrophoresis of Bacillus cereus Group Strains Paul Drean and Edward M. Fox Abstract Pulsed-Field Gel Electrophoresis (PFGE) subtyping has been used extensively to characterize various bacterial species and to facilitate comparative analysis of geographically diverse populations. To this end, standardized protocols for many different genera and species have been developed, particularly through the PulseNet platform. The Bacillus cereus group of bacteria includes a diverse species set, which are of particular importance in food safety as both human pathogens and spoilage organisms. The application of techniques to differentiate strains of B. cereus can be utilized to assist in both disease outbreak investigations, and also in strategies to monitor and control the organism in food production environments. This chapter describes a PFGE method, which may be applied to differentiate B. cereus strains. Keywords Pulse-field gel electrophoresis, Bacillus cereus group

1

Introduction The B. cereus group are facultative anaerobic, spore-forming bacteria. This group of bacteria comprises a set of closely related organisms, including B. cereus, B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. anthracis, and B. cytotoxicus. Differentiation of these species is complex; with high syntenty among the grouping, phenotypic traits are central to their identification [1, 2]. This includes characteristics such as colony morphology and growth temperature range [1]. Understanding the biology of this group of organisms can be complex, as differentiation between the species B. cereus, as opposed to the B. cereus group, is not always clear in studies. This is exemplified in the ISO standard method for isolation of “B. cereus,” which in fact does not differentiate the members of the B. cereus group; though it should be noted that B. mycoides and B. pseudomycoides display rhizoid colony morphology [3]. Differentiation of species, however, is of critical importance in understanding associated health risks, with B. anthracis strains capable of producing a particularly potent toxin [4].

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_7, © Springer Science+Business Media New York 2015

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Commonly associated with soil, the B. cereus group species can cross into food production chains though a variety of routes— including direct contamination of cultivated fruit and vegetables, or ingestion by animals leading to subsequent contamination of related food products or environments. Contaminated food products can subsequently cause human disease, if sufficient numbers and conditions allow production of toxin by associated B. cereus group strains. A wide range of toxins may be produced by strains, including cerulide, which leads to an emetic illness, and haemolysin BL and nonhaemolytic enterotoxin, which can cause diarrhoeal disease [1, 5]. Contamination of food products may also lead to spoilage of the food product itself, making it unfit for human consumption [2, 5]. PFGE has been applied to many organisms to gain insights into population dynamics [6–8]. The PFGE method presented here may be used as a highly discriminatory tool for strain differentiation of members of the B. cereus group; however, it is not suitable as a tool for species differentiation.

2

Materials Prepare all solutions using ultrapure water (prepared with Reagent Grade Type 1 ultrapure water to attain a sensitivity of 18.2 MΩ cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at 18–25 °C (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.

2.1 Growth of the Cultures

1. Brain Heart Infusion Agar (BHIA) plates.

2.2 Cell Suspension and Casting Plugs

1. Sterile TE Buffer (10 mM Tris:1 mM EDTA, pH 8.0): Add 10 mL of 1 M Tris, pH 8.0 to 2 mL of 0.5 M EDTA, pH 8.0 and dilute to 1,000 mL with sterile ultrapure water.

2. Sterile loops.

2. Lysozyme (20 mg/mL) stock solution. Weigh 100 mg lysozyme, add 5 mL TE Buffer, swirl to mix and aliquot 250 μL volumes into sterile tubes, and freeze at −20 °C for future use. Once thawed, discard any unused lysozyme reagent. 3. Proteinase K (20 mg/mL) stock solution. The stock solution of Proteinase K can be prepared by weighing 100 mg of powder into 5 mL sterile ultrapure water. Aliquot 250 μL volumes into sterile 1.5 mL tubes and freeze at −20 °C for future use. Once thawed, discard any unused Proteinase K reagent. 4. Prepare 1 % SeaKem Gold agarose (SKG) in TE Buffer for PFGE plugs: Weigh 0.1 g SKG agarose into a flask or Schott bottle; add 10 mL TE Buffer; swirl gently to disperse the agarose, loosen the cap, and microwave for 30 s; mix gently and

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repeat for 10 s intervals until the agarose is completely dissolved. Place the agarose in a water bath set to 55–60 °C and equilibrate until ready to use. 5. Sterile polyester-fiber or cotton swabs. 6. Spectrophotometer and cuvettes. 7. 50-well disposable plug mold or 10-well reusable plug mold. 1. Cell Lysis Buffer (CLB; 50 mM Tris:50 mM EDTA, pH 8.0 + 1 % Sarcosyl): Add 50 mL of 1 M Tris, pH 8.0 to 100 mL of 0.5 M EDTA, pH 8.0; add 100 mL of 10 % Sarcosyl (NLauroylsarcosine, Sodium salt) and dilute to 1,000 mL with sterile Ultrapure water (see Note 1).

2.3 Lysis of Cells in Agarose Plugs

2. Proteinase K (20 mg/mL) (see Subheading 2.2, item 3). 2.4 Washing of Agarose PLUGS

1. Sterile water. 2. Sterile TE Buffer (see Subheading 2.2, item 1). 3. Spatula. 1. “3.1 Buffer 10×” or “CutSmart Buffer 10×” (adjust as appropriate to the brand of restriction enzyme in use).

2.5 Restriction Digestion of DNA in Agarose Plugs

2. NotI, SbfI, or XbaI restriction enzymes. 3. Sterile blade. 4. Glass slide or petri dish. 5. Spatula. 1. Thiourea, 500 mM: Dissolve 3.806 g Thiourea to a final volume of 100 mL in ultrapure water.

2.6 Casting of the Agarose Gel

2. Gel and Electrophoresis Running Buffer (0.5× Tris-Borate EDTA Buffer) (TBE, Table 1): Add 2.2 mL of 500 mM Thiourea and 110 mL of 10× TBE stock solution into 2,090 mL sterile ultrapure Water.

Table 1 Electrophoresis running buffer (0.5×TBE) Reagent

Volume (mL)

500 mM thiourea

2.2

10× TBE

110 a

a

Sterile ultrapure water

2,090

Total volume of 0.5× TBE

2,200

Any water suitable for molecular biology can be used

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3. 1 % SKG agarose in 0.5× TBE: Weigh the appropriate amount of SKG into a screw cap flask (or Schott bottle): –

Mix 1.0 g agarose with 100 mL 0.5× TBE for 14 cm-wide gel form.

–

Mix 1.5 g agarose with 150 mL 0.5× TBE for 21 cm-wide gel form.

–

Add the appropriate amount of 0.5× TBE; swirl gently to disperse the agarose, loosen the cap, and microwave for 60 s; mix gently and repeat for 15 s intervals until the agarose is completely dissolved. Return the flask (or Schott bottle) to 55–60 °C water bath until ready to use.

4. Gel comb. 2.7 Electrophoresis Conditions

1. PFGE equipment CHEF Mapper, CHEF-DR III, or CHEF-DR II. 2. Cooling module for PFGE system. 3. 2 L of 0.5× TBE buffer containing 500 μM Thiourea (Table 1).

2.8 DNA Size Markers

1. Salmonella enterica subsp. enterica ser. Braenderup H9812 marker digested with XbaI restriction enzyme in CutSmart Buffer. (The buffer used should be suitable for the particular XbaI brand of enzyme used.) 2. Saccharomyces cerevisiae Chef DNA Size marker.

2.9 Staining and Imaging of the Agarose Gel

3

1. GelRed® Nucleic Acid Gel Stain 10,000× in water (see Note 2). 2. Imaging equipment Gel Doc 1000 or equivalent. 3. BioNumerics Software.

Methods Carry out all procedures at 18–25 °C unless otherwise specified.

3.1 Grow of the Cultures

1. Streak an isolated colony from test cultures onto a BHIA plate (or comparable nonselective media) for confluent growth (see Note 3). 2. Incubate the cultures at 30 °C for 16–18 h.

3.2 Preparation of DNA Plugs

1. Turn on a shaking water bath or incubator with a shaker, set to 55 °C. 2. Set the spectrophotometer to 600 nm wavelength and prepare the blank with sterile TE Buffer. 3. Label suitable tubes with the culture numbers and transfer 2 mL of TE Buffer into each tube. Use a sterile polyester-fiber

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or cotton swab that has been moistened with sterile TE to remove colonies from the agar plate; suspend the cells in TE Buffer by spinning the swab gently so that the cells will be evenly dispersed and the formation of aerosols is minimized (see Note 4). 4. Adjust the concentration of cell suspensions to an OD600nm of 1.00 by diluting with sterile TE Buffer or by adding additional cells, as required. 3.3 Casting the Plugs

1. Label the wells of the PFGE plug molds with the culture numbers. If disposable plug molds are used, seal the bottom of the wells with a strip of sticky tape on the lower part of the reusable plug mold before labeling the wells. 2. Transfer 400 μL of adjusted cell suspensions to labeled 1.5 mL tubes. 3. Add 20 μL of thawed lysozyme (20 mg/mL) from the stock solution to each tube and mix by gently pipetting five to ten times. 4. Place the tubes into the 55 °C shaking water bath or incubator with a shaker for 2 h and discard any unused thawed lysozyme solution. 5. Add 20 μL of Proteinase K (20 mg/mL) from the stock solution to each tube and mix gently with pipette tip. 6. Add 400 μL melted 1 % SKG agarose to 400 μL of cell suspension; mix by gently pipetting the mixture up and down approximately five times (overpipetting can cause DNA shearing). Maintain the temperature of the melted agarose by keeping the flask in a beaker of warm water (55–60 °C). 7. Immediately dispense part of the mixture into appropriate well(s) of the plug mold. Do not allow air bubbles to form. Multiple plugs of each sample can be made from these amounts of cell suspension and agarose, which are useful if the repeated testing is required. Allow the plugs to solidify at 18–25 °C for 10–15 min. They can also be placed in the refrigerator (4 °C) for 5 min (see Note 5).

3.4 Lysis of Cells in the Agarose Plugs

1. Label 15 mL tubes with culture numbers. 2. Calculate the total volume of CLB-Proteinase K for a final concentration of 0.1 mg/mL Proteinase K: ●

●

5 mL CLB is needed per tube. 25 μL Proteinase K stock solution (20 mg/mL) is needed per tube of the CLB.

3. Prepare the master mix by measuring the correct volume of CLB and Proteinase K into an appropriate size test tube or flask and mix well.

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4. Add 5 mL of CLB-Proteinase K to each labeled 15 mL tube. 5. Trim the excess agarose from top of the plugs with a sterile blade. Open the reusable plug mold and transfer the plugs from the mold with a 6-mm wide spatula to appropriately labeled tubes. If disposable plug molds are used, remove the sticky tape from the bottom of the plug mold and push out the plug(s) into appropriately labeled tube, note that two plugs (reusable molds) or three to five plugs (disposable molds) of the same strain can be lysed in the same 15 mL tube. Be sure the plugs are covered with buffer. 6. For the reusable plug mold, place both sections of the plug mold, spatulas, and scalpel in 90 % ethanol, 1 % Lysol/Amphyll, or other suitable disinfectant. Soak them for 15 min before washing them thoroughly with water. Discard disposable plug molds or disinfect them in 90 % ethanol for 30–60 min if they will be washed and reused. 7. Place the tubes in a rack and incubate at 55 °C in a water bath or incubator with a shaker overnight (160 rpm). If lysing in a water bath, be sure water level is above the level of CLB in the tubes. 8. Preheat enough sterile ultrapure water to 50 °C so that the plugs can be washed twice with 10–15 mL water (200–300 mL for ten tubes). 3.5 Washing of Plugs After Cell Lysis

1. Remove the tubes from the water bath or incubator and carefully pour off the CLB into an appropriate discard container; plugs can be held within the tubes by using a screened cap or spatula. It is important to remove all of the liquid. 2. Add 10–15 mL sterile ultrapure water that has been preheated to 50 °C to each tube and shake the tubes at 160 rpm in a 50 °C water bath or incubator with a shaker for 10–15 min. 3. Pour off the water from the plugs and repeat the wash step with preheated water (step 2) one more time. 4. Preheat enough sterile TE Buffer (as outlined in Subheading 2.2, item 1) in a 50 °C water bath so that the plugs can be washed four times with 10–15 mL TE (400–600 mL for ten tubes). Pour off the water, add 10–15 mL preheated (50 °C) sterile TE Buffer, and shake the tubes at 160 rpm in a 50 °C water bath or incubator with a shaker for 10–15 min. 5. Pour off the TE and repeat the wash step with preheated TE three more times. Decant the last wash buffer. Continue with step 1 in Subheading 3.6 or store the plugs in TE Buffer at 4 °C until needed. (Plugs are stable for at least 4 weeks.) If restriction digestion is to be done on the same day, complete steps 1–3 of Subheading 3.6 during the last TE wash step for optimal use of time.

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Table 2 Prerestriction digest equilibrium buffer (1×) Reagent

μL/plug slice

μL/15 plug slices

Sterile ultrapure watera

180

2,700

3.1 buffer 10× or CutSmart buffer 10×

20

300

200

3,000

Total volume a

Any water suitable for molecular biology can be used

3.6 Restriction Digestion of DNA in Agarose Plugs

1. Turn on a water bath to 37 °C. 2. Label 1.5 mL tubes with culture numbers. Label 3 for S. ser. Braenderup H9812 standards. 3. Dilute 3.1 Buffer 10× or CutSmart Buffer 10× to a final concentration of 1× with molecular-biology grade water as appropriate, according to Table 2. This will be used as the equilibration buffer. 4. Carefully remove a plug from the TE with a spatula and place it on a large glass slide (or sterile disposable Petri dish). 5. Cut a 2.0–2.5 mm-wide slice from the DNA plug with a sterile blade to ensure all rough edges are removed, and transfer to a tube containing the appropriate 200 μL diluted 3.1 Buffer (1×) for NotI restriction or CutSmart Buffer (1×) for SbfI or XbaI restriction, ensuring the plug slice is under the buffer (use the same buffer that is used for the enzymatic restriction). 6. Replace the rest of the plug in the original tube that contains 5 mL TE Buffer and store it at 4 °C. 7. Cut three 2.0 mm-wide slices from the plug of the S. ser. Braenderup H9812 standard and transfer them to tubes with 200 μL diluted CutSmart Buffer, ensuring the plugs are under buffer. Again, replace the rest of the plug in the original tube that contains 5 mL TE Buffer and store at 4 °C. 8. Equilibrate by incubating in the buffer at 18–25 °C for 15–20 min. 9. After incubation, remove the TE Buffer from the plug slice by pipetting with a 200–250 μL tip. Care is needed to ensure that the plug slice remains intact. 10. For the B. cereus DNA plugs, dilute 3.1 Buffer 10× 1:10 with molecular grade water and add NotI restriction enzyme (40 U/ sample) or dilute CutSmart Buffer 10× 1:10 with molecular grade water and add SbfI restriction enzyme (40 U/sample) according to Table 3 or 4. Keep the restriction enzyme on ice at all times.

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Table 3 Restriction digestion of B. cereus DNA plugs with Not I Reagent

μL/plug slice

μL/12 plug slices

Sterile ultrapure watera

176

2,112

4

48

20

240

200

2,400

NotI enzyme (10 U/μl) 3.1 buffer (10×) Total volume a

Any water suitable for molecular biology can be used

Table 4 Restriction digestion of B. cereus DNA plugs with Sbf I Reagent

μL/plug slice

μL/12 plug slices

Sterile ultrapure watera

176

2,112

SbfI enzyme (10 U/μL)

4

48

CutSmart buffer (10×)

20

240

200

2,400

Total volume a

Any water suitable for molecular biology can be used

Table 5 Restriction digestion of Salmonella marker plugs with Xba I Reagent

μL/plug slice

μL/3 plug slices

Sterile ultrapure watera

175

525

5

15

20

60

200

600

XbaI enzyme (10 U/μL) CutSmart buffer (10×) Total volume a

Any water suitable for molecular biology can be used

11. For the Salmonella marker DNA plugs, dilute CutSmart Buffer 10× 1:10 with molecular grade water and add XbaI restriction enzyme (40 U/sample) according to Table 5. 12. Add 200 μL of restriction enzyme mixture to each tube containing the DNA plug slice, as appropriate. Close the tube and mix by tapping gently, ensuring that the plug slices are under enzyme mixture. 13. Incubate the sample at 37 °C for 2 h for NotI, SbfI, and XbaI (see Note 6).

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3.7 Casting Agarose Gel and Loading DNA Plugs

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1. Ensure the water bath is equilibrated to 55 °C. 2. Clean the PFGE gel tank with the preheated (55 °C) pure deionized water and circulate for a minimum of 30 min ( see Note 7). 3. Drain the water from the PFGE gel tank. 4. Add 2 L of the 0.5× TBE containing Thiourea to the PFGE gel tank ensuring that there are no air bubbles and that the unit is level, then close the cover unit. 5. Turn on the cooling module (14 °C), power supply, and pump (setting of ~70 for a flow of 1 L/min). 6. Remove the restricted plug slices from the 37 °C water bath (or from the fridge). Remove the enzyme/buffer mixture and add 200 μL 0.5× TBE. Allow to stand at 18–25 °C for 5–10 min. 7. Cut three 2 mm slices of S. cerevisiae DNA Size Chef Marker using the same method as for the previous plugs. Put the slices in three different marked 1.5 mL tubes and add 200 μL 0.5× TBE. Allow to stand at 18–25 °C for 5–10 min. 8. Remove the plug slices from the tubes and lay them gently on a tissue to remove any excess buffer. Put the comb on the bench top and load the plug slices on the bottom of the comb teeth with the S. ser. Braenderup H9812 and S. cerevisiae standards on teeth (lanes) 1, 5, and 10 of a 14 cm comb, or lanes 1, 8, and 15 of a 21 cm comb. Load the two marker plugs one directly above the other on the comb teeth. This will result in the size marker profile as shown in Fig. 1, following electrophoresis. Load the samples on the remaining teeth. 9. Confirm that the plug slices are correctly aligned on the bottom of the teeth. Ensure that there are no air bubbles. 10. Allow the plug slices to air dry on the comb for 5 min. 11. Place the gel platform on a leveling table and adjust until perfectly leveled before pouring the gel. Position the comb holder so that the front part (side with small metal screws) and teeth face the bottom of the gel and the bottom edge of the comb is 2 mm above the surface of the gel platform, and carefully pour the agarose (cooled to 55–60 °C) into the gel mold. 12. Remove the comb after the gel solidifies (this will take approximately 20 min). 13. Unscrew and remove the end gates from gel mold; remove the excess agarose from the sides and bottom of casting platform with a tissue. 14. Ensure the buffer in the gel tank has reached 14 °C (see Subheading 3.7, step 5). Keep the gel on the casting platform and carefully place the gel inside black gel frame in the electrophoresis chamber. Close the cover of the chamber.

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Fig. 1 DNA marker used for B. cereus group PFGE analysis. This marker is a combination of S. enteric subsp. enterica ser. Braenderup H9812 XbaI restricted DNA, and a S. cerevisiae CHEF DNA Size marker. This ensures that all high molecular weight B. cereus DNA fragments can be included in the analysis

3.8 Electrophoresis Conditions

1. Set up the corresponding conditions for your electrophoresis equipment: ●

Conditions for CHEF Mapper: ●

Auto algorithm.

●

20 kb: low MW.

●

700 kb: high MW.

●

●

●

●

Select default values except where noted by pressing “enter.” Set run time to 21 h (see Note 8). Default values: Initial switch time = 5.0 s; Final switch time = 80.0 s.

Conditions for CHEF-DR III: ●

Initial switch time: 5.0 s.

●

Final switch time: 80.0 s.

Pulsed-Field Gel Electrophoresis of Bacillus cereus Group Strains

●

●

Voltage: 6 V.

●

Included angle: 120°.

●

Run time: 21 h (see Note 8).

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Conditions for CHEF-DR II: ●

Initial A time: 5.0 s.

●

Final A time: 80.0 s.

●

Start ratio: 1.0 (if applicable).

●

Voltage: 200 V.

●

Run time: 21 h (see Note 8).

2. Start the run. Make note of the initial milliamp (mA) reading on the instrument. The initial mA should be between 110 and 150 mA. A reading outside of this range may indicate that the 0.5× TBE buffer was prepared improperly and the buffer should be remade. 1. When electrophoresis run is over, turn off the equipment and remove the gel.

3.9 Staining and Imaging of the Agarose Gel

2. Dilute 30 μL GelRed® (see Note 2) with 300 mL of ultrapure water and stain the gel with this solution for 30 min in a covered container. 3. Destain the gel in approximately 300 mL of ultrapure water for 60–90 min, changing the water every 20 min. 4. Capture the image on Gel Doc 1000 or equivalent documentation system. If the background interferes with resolution, destain for an additional 30–60 min. Figure 2 shows an example of PFGE fingerprint profiles from different B. cereus group strains. 5. Follow the directions given with the imaging equipment to save gel image as an *.tif file. Also, save the image using the file-type utilized by the software. The *.tif file can be utilized for analysis with BioNumerics software or other suitable software programs (see Note 9).

Optimisation & Tolerance: 1% 50

60

70

80

90

PFGE - NotI

PFGE – SbfI

Isolate

Species___________

100

Bc14-006

. pseudomycoides B.

Bc14-026

. weihenstephanensis B.

Bc13-003

B. . cereus

Bc13-017

B. . thuringiensis

Bc14-011

B. mycoides

Fig. 2 PFGE similarity analysis for representative species of the B. cereus group

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6. Drain the buffer from the electrophoresis chamber and discard it. Rinse the chamber with 2 L of preheated (50 °C) molecular grade water and flush the lines with water by letting the pump run for 15–20 min before draining the water from the chamber and hoses.

4

Notes 1. To dissolve the Sarcosyl easily, add 10 g of Sarcosyl directly to the Cell Lysis buffer when preparing the reagent. This replaces adding 100 mL of a 10 % Sarcosyl solution, as Sarcosyl solution can be difficult to dissolve when prepared at 10 %. 2. Other staining solutions can be used, such as ethidium bromide or SYBR Safe DNA gel Stain 10,000× concentrate in DMSO. 3. It is recommended that a storage vial of each culture be created. This will ensure that the same colony can be retested if necessary. 4. Keep cell suspensions on ice if you have more than six cultures to process or refrigerate cell suspensions if you cannot adjust their concentration immediately. 5. Five plugs can be made from these amounts of cell suspension and agarose. The generation of cell suspension and the subsequent casting of the plugs should be performed as rapidly as possible in order to minimize premature cell lysis. If a large number of samples are being prepared, it is recommended that they be processed in batches of around ten samples at a time. Once the first batch of isolates are in the cell lysis incubation, then start preparing the cell suspensions for the next group of samples and so on. All batches can be lysed and washed together, since additional lysis time will not affect the initial batches. 6. SbfI is recommended as the secondary enzyme for analysis of Bacillus cereus group isolates. The use of a secondary enzyme is useful in situations where the PFGE patterns obtained with the primary enzyme are indistinguishable (e.g., for the identification of highly related clonal groups). Check the conditions for the restriction enzymes as different suppliers may specify different temperatures. 7. Steps 2 and 3 of Subheading 3.7 may be skipped if the PFGE gel tank has been recently cleaned as described in these steps. 8. The electrophoresis running times recommended above are based on the equipment and reagents used at the Centers for Disease Control and Prevention. Run times may be different in your laboratory and will have to be optimized for your gels so

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that the lowest band in the S. ser. Braenderup H9812 standard migrates within 1.0–1.5 cm of the bottom of the gel. 9. When using the Reference Marker as shown in Fig. 1, only use the bands that are indicated in Fig. 1. This will ensure the best resolution of the B. cereus group strain PFGE fingerprints. References 1. Ceuppens S, Boon N, Uyttendaele M (2013) Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles. FEMS Microbiol Ecol 84:433–450 2. Tourasse NJ, Helgason E, Økstad OA, Hegna IK, Kolstø A-B (2006) The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol 101:579–593 3. International Organization for Standardization (2004) Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of presumptive Bacillus cereus - Colony-count technique at 30 degrees C. ISO 7932:2004, 1–13 4. Tournier JN, Rossi-Paccani S, Quesnel-Hellmann A, Baldari CT (2009) Anthrax toxins: a weapon to systematically dismantle the host immune defenses. Mol Aspects Med 30:456–466

5. Arslan S, Eyi A, Küçüksari R (2014) Toxigenic genes, spoilage potential, and antimicrobial resistance of Bacillus cereus group strains from ice cream. Anaerobe 25:42–46 6. Lynch MJ, Fox EM, O’Connor L, Jordan K, Murphy M (2012) Surveillance of verocytotoxigenic Escherichia coli in Irish bovine dairy herds. Zoonoses Public Health 59:264–271 7. Fox EM, DeLappe N, Garvey P, Cormican M, Leonard N, Jordan K (2012) PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland. J Med Microbiol 61:540–547 8. Stessl B, Fricker M, Fox E, Karpiskova R, Demnerova K, Jordan K, Ehling-Schulz M, Wagner M (2014) Collaborative survey on the colonization of different types of cheeseprocessing facilities with Listeria monocytogenes. Foodborne Pathog Dis 11:8–14

Chapter 8 Pulsed-Field Gel Electrophoresis of Staphylococcus aureus George R. Golding, Jennifer Campbell, Dave Spreitzer, and Linda Chui Abstract For many bacterial pathogens, including Staphylococcus aureus, pulsed-field gel electrophoresis (PFGE) is a molecular typing method widely used in surveillance and epidemiological investigations. The general principle of PFGE involves creating large DNA fragments from intact bacterial chromosomes using rare cutting restriction endonucleases. These large DNA fragments are successfully separated in an agarose gel by alternating the direction of the electrical fields over a prolonged period of time. The resulting DNA banding patterns in the gel create a “DNA fingerprint,” which can then be used to discriminate clonal relatedness of isolates based on set interpretation guidelines. Standardization of protocols has greatly enhanced the reproducibility of PFGE between labs, enabling national surveillance and further molecular epidemiological studies of S. aureus. Keywords Pulsed-field gel electrophoresis, PFGE, Staphylococcus aureus, Methicillin-resistant, MRSA, Molecular typing, Epidemiology

1

Introduction Staphylococcus aureus is a leading nosocomial and communityacquired bacterial pathogen. Over the past 50 years, the emergence of methicillin-resistant S. aureus (MRSA) has had a dramatic impact on the health care systems worldwide, which is further compounded over the last two decades with the emergence of community [1, 2] and livestock-associated MRSA [3, 4] strain types. With increasing rates of MRSA infections and new community sources, it has become important to quickly distinguish strains of MRSA for the purpose of epidemiological investigation and infection control. While a variety of strain typing methods have been used over the years, including spa [5, 6] and multilocus sequence typing (MLST) [7], pulsed-field gel electrophoresis (PFGE) is still considered the “gold standard” for determining the degree of relatedness between isolates of MRSA in outbreak investigations.

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_8, © Springer Science+Business Media New York 2015

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PFGE of S. aureus requires the isolation of intact bacterial chromosomes, which are subsequently digested into large DNA fragments using the rare cutting restriction endonuclease SmaI. Since high molecular weight DNA is easily cleaved, care must be taken to minimize shearing and degradation throughout the procedure. To achieve this, all manipulations including cell lysis, protein removal, and restriction digestions are performed in agarose plugs, which are then loaded into the wells of an agarose gel. However, conventional electrophoresis is only able to separate DNA fragments up to 20–50 kb. To circumvent this, Schwartz and Cantor [8] demonstrated that by alternating the direction of the electrical field DNA fragments of up to ~2 Mb could be resolved. Since then, a variety of alternative apparatuses and methods have been developed [9–12]. Contour-clamped homogeneous electric field electrophoresis (CHEF) is the most commonly used PFGE technique [12]. The advantage of the CHEF apparatus is that it is capable of separating large DNA molecules in straight lines by generating homogeneous electric fields using 24 electrodes positioned in a closed hexagonal contour around the gel. Field reorientation is achieved by alternating the voltages of the electrodes at a 120o fixed angle for prolonged period of time. Following electrophoresis, the gels are stained and the resulting banding patterns are captured by an imaging system and analyzed using software programs such as BioNumerics. Standardization of protocols has greatly enhanced the reproducibility of PFGE [13], enabling collaborative national/international surveillance systems [2, 14, 15], and with set interpretation guidelines [16] has been an invaluable molecular epidemiological tool for outbreak studies of S. aureus.

2

Materials

2.1 Culture and Agarose Plug Preparation

1. 37 °C incubator. 2. Salmonella Braenderup H9812 as molecular weight standard. 3. Tryptic soy agar (TSA) plates containing 5 % sheep’s blood. 4. Water baths set at 37 and 55 °C. 5. Bio-Rad PFGE agarose. 6. Cell suspension buffer (CSB), 10 mM Tris–HCl pH 7.2, 20 mM NaCl, 50 mM EDTA. Mix 10 mL of 1 M Tris–HCl, pH 7.2; 20 mL of 1 M NaCl; and 100 mL of 0.5 M EDTA, pH 8.0. Dilute to 1 L using 18 MΩ dH2O. Autoclave to sterilize and store up to 6 months at 18–25 °C. 7. Microwave. 8. Sterile, 1.5 mL microfuge tubes. 9. Plug molds (Bio-Rad or equivalent).

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10. 5 mL tubes. 11. Sterile cotton swabs or equivalent. 12. Turbidity meter. 13. Lysostaphin. 2.2 Cell Lysis in Agarose Plugs

1. Sterile, 1.5 mL microfuge tubes. 2. Cell Lysis Buffer (CLB), 10 mM Tris–HCl pH 7.2, 50 mM NaCl, 50 mM EDTA, 0.2 % deoxycholate, 0.5 % N-Laurylsarcosine. Mix 10 mL of 1 M Tris–HCl, pH 7.2, 50 mL of 1 M NaCl, 100 mL of 0.5 M EDTA, pH 8.0, 2 g of deoxycholate, and 5 g of N-Lauryl Sarcosine Sodium salt. Dilute to 1 L using 18 MΩ dH2O. Filter sterilize using 0.22 μm filter and store up to 6 months at 18–25 °C. 3. Water baths set at 37 and 55 °C. 4. Proteinase K. 5. Proteinase K buffer, 250 mM EDTA pH 9.0, 1 % N-Laurylsarcosine. Mix 500 mL of 0.5 M EDTA, pH 8.0 and 10 g N-Lauryl Sarcosine Sodium salt. Dilute to 1 L using 18 MΩ dH2O. Filter sterilize using 0.22 μm filter and store up to 6 months at 18–25 °C.

2.3

Plug Washing

1. TE Buffer, 10 mM Tris–HCl pH 8.0, 1 mM EDTA. Mix 10 mL of 1 M Tris–HCl, pH 8.0 and 0.2 mL of 0.5 M EDTA, pH 8.0. Dilute to 1 L using 18 MΩ dH2O. Autoclave to sterilize and store up to 6 months at 18–25 °C. 2. Gyrating shaker. 3. 4 °C refrigerator.

2.4 Restriction Digestion of DNA in Agarose Plugs

1. Sterile, 0.6 mL microfuge tubes. 2. Sterile spatula and scalpel. 3. Sterile cutting surface. 4. 1× TE buffer. 5. 10× Restriction Buffer A and Buffer H supplied with the restriction enzyme. 6. SmaI and XbaI restriction enzyme, 40 U/μL. 7. Incubators set at 37 and 25 °C. 8. 70 % ethanol.

2.5 PFGE Electrophoretic Run Conditions

1. CHEF DRIII PFGE System. 2. CHEF gel casting platform and accessories. 3. Commercial 5× TBE.

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2.6 Casting Agarose Gels and Loading Restricted Plugs into Wells

1. Bio-Rad PFGE Agarose.

2.7 Staining and Documentation of the Gel

1. Ethidium Bromide, 10 mg/mL.

2. Commercial 5× TBE. 3. Water bath set at 55 °C. 4. Kimwipes or equivalent lint free towels.

2. Sterile, 18 MΩ ddH2O. 3. Gyrating shaker or equivalent. 4. Tupperware containers for staining and destaining of gels. 5. Gel Doc 2000 or equivalent.

2.8

3

Gel Analysis

1. BioNumerics version 5.1 or equivalent.

Methods

3.1 Culture and Agarose Plug Preparation

1. To prepare from an agar plate, streak the isolate out on a TSA agar plate containing 5 % sheep’s blood and incubate at 37 °C overnight (16–18 h). 2. Turn on the 55 and 37 °C water baths. 3. Prepare 1 % PFGE agarose in 20 mL of CSB. Dissolve the agarose in a microwave and mix gently. Before use, place it in a 55 °C water bath for 10 min to equilibrate (see Note 1). 4. Label 1.5 mL microfuge tubes for each test strain. 5. Prepare and label plug molds for each test isolate. 6. Label 5 mL tubes and transfer 1.5 mL of CSB into the labeled tubes. Use a sterile polyester-fibre or cotton swab to remove some of the growth from the agar plate and resuspend the cells in CSB by rotating the swab gently inside the tube. 7. Adjust the concentration of the cell suspensions using a Turbidity Meter to 0.48–0.52 by diluting with sterile CSB or by adding additional cells (see Note 2). 8. Transfer 150 μL of each cell suspension into labeled 1.5 mL tubes. 9. Add 2 μL of 1 mg/mL lysostaphin and mix gently by pipetting. 10. Transfer 150 μL of melted 1 % PFGE agarose to 150 μL of each cell suspension (see Note 3). 11. Pipette the agarose mixture into two wells of the labeled disposable plug mold. 12. Allow the plugs to solidify at 18–25 °C (10 min) or at 4 °C (5 min).

Pulsed-Field Gel Electrophoresis of Staphylococcus aureus

3.2 Cell Lysis in Agarose Plugs

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1. Label a 1.5 mL microfuge tube for each test strain and transfer 500 μL of CLB to the tubes. 2. Transfer two solidified plugs carefully from the disposable plug mold into appropriately labeled tubes and incubate for 30 min at 37 °C. 3. Remove the tubes from the 37 °C water bath and carefully aspirate off the lysis buffer. 4. Add 500 μL of the Proteinase K buffer containing 2.5 μL of 20 mg/mL Proteinase K to each tube. 5. Transfer the tubes to the 55 °C water bath and incubate for 40 min or overnight if required.

3.3

Plug Washing

1. Prepare enough sterile 1× TE Buffer for seven washes (two rinses, five washes). 2. Remove the tubes from the water bath and using a pipettor aspirate off the Proteinase K solution. 3. Rinse the plugs twice using 1.5 mL of 18–25 °C 1X TE. Hold the cap of the tube open slightly and invert to empty it, being careful not to pour out the plugs along with the wash buffer. 4. Fill the tubes again with 1× TE and place the tubes on rotator/ shaker for 5 min. Repeat this step once more. The next three washes will be carried out in intervals of 10, 15, and 20 min. 5. Store the tubes at 4 °C for up to 6 months.

3.4 Restriction Digestion of DNA in Agarose Plugs

1. Label one 0.6 mL tube for each sample and S. ser. Braenderup H9812 standard plug slice that is going to be digested (see Note 4). 2. Carefully remove one agarose plug from the storage tube with a sterile spatula and place it on a sterile cutting surface (see Note 5). 3. Place the remaining sample and control plugs into 1X TE buffer and store at 4 °C. 4. Using a scalpel, cut off approximately 1/4 of the plug lengthwise (2.0–2.5 mm slice) and transfer it to the 0.6 mL labeled tube and return the rest of the plug into the 1.5 mL storage tube. Repeat this step for the remaining samples (see Note 6). 5. Equilibrate the plugs by adding 150 μL of 1× buffer A to the tubes containing the 1/4 slice of sample plug and add 150 μL of 1× buffer H to the tubes containing the 1/4 slice of standard plugs (see Note 7). 6. Remove the buffer after 10–15 min with a micropipettor, taking care not to damage or remove the plug from the tube. 7. Prepare fresh 1× buffer A (100 μL/plug slice), add 40U SmaI enzyme (40 U/μL) per 100 μL buffer.

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8. Mix well and aliquot 100 μL of the buffer A/SmaI mixture into each equilibrated sample tube. 9. Prepare fresh 1× buffer H, add 40 U XbaI enzyme (40 U/μL) per 100 μL buffer. Mix well and aliquot 100 μL of the buffer H/XbaI mixture into each equilibrated standard tube. 10. Digest the SmaI sample plugs at 25 °C for 2 h minimum or overnight. 11. Digest the XbaI control plugs at 37 °C for 2 h minimum or overnight. 3.5 PFGE Electrophoretic Run Conditions

1. Place a 10 × 10 cm black gel frame into the CHEF chamber. 2. Add 2.0–2.5 L of fresh 0.5× TBE to the chamber. 3. Close the chamber cover and turn on the power supply, the circulation pump, and the cooling module. Set the cooling module to 14 °C. 4. Program the following run conditions on CHEF DRIII unit (see Note 8): Initial A switch time

5.3 s

Final A switch time

34.9 s

Voltage

6 V/cm

Included angle

120

Start ratio

1.0

Run time

20 h

5. Allow the buffer in the chamber to cool for approximately 30 min prior to running the gel and ensure the buffer temperature is 14 °C. 3.6 Casting Agarose Gels and Loading the Restricted Plugs into Wells

1. Prepare 100 mL of 1 % PFGE agarose in 0.5× TBE. 2. Place the melted agarose in the 55 °C water bath to equilibrate for 20 min or until ready to pour. 3. Assemble a 14 × 14 cm gel casting PFGE form on a level surface. Place a 15-well comb (0.75 cm teeth) into the tray to make the wells. 4. Place the comb on the holder and adjust it so that the comb’s teeth touch the bottom of the gel casting tray. 5. Aspirate all the 1× restriction enzyme buffer/enzyme from the restricted sample and standard plug slices with a pipettor taking care not to damage the plugs. 6. Load the digested plug slices on the comb. Use a kimwipe to remove excess moisture. Push the plugs down so that they contact the bottom of casting tray and let them dry for 5 min.

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7. Slowly pour the melted agarose into a corner of the PFGE casting form (see Note 9). 8. Allow the gel to solidify at 18–25 °C (approximately 30–45 min) and then remove the comb and disassemble the casting tray. 9. Transfer the gel to the CHEF chamber and check all run conditions and ensure that the buffer temperature is 14 °C. 10. Press start. 3.7 Staining and Documentation of the Gel

1. When the run is complete, turn off the equipment and drain the buffer from the chamber. Rinse the chamber with water if the unit is not to be used immediately. 2. Transfer the gel into the ethidium bromide staining solution (EtBr) (0.5 mg/L) and stain on a gyrating shaker for at least 30 min. 3. Transfer the gel to a destaining container containing 18 MΩ ddH2O and destain as long as necessary (30 min to 3 h) (see Note 10). 4. View the gel using an imaging software. Photograph and save the gel image as a .tiff file for further software analysis.

3.8

Gel Analysis

1. Open BioNumerics. 2. The gel TIFF image is imported and processed through the following four steps as outlined in the BioNumerics manual: (1) import and convert the TIFF image to gel strip and define the lanes, (2) edit the gel tone curve to optimize the image, (3) normalization against the S. Braenderup H9812 standard, and (4) define the gel bands of the test isolates on the normalized gel strips. 3. For creating dendrograms, the similarity coefficient should be band based using the Dice coefficient and the dendrogram type should be set to unweighted pair group method using averages (UPGMA) with a position tolerance of 1.0 % and an optimization of 1.0 %. A cluster analysis of some of the major MRSA clones in Canada is shown in Fig. 1.

4

Notes 1. Do not melt the agarose more than three times. 2. A uniform concentration of cells is required for standardized amounts of DNA. 3. Mix gently to prevent shearing of DNA and to ensure that no air bubbles are introduced. The temperature of the melted agarose can be maintained by keeping the media in a beaker of warm water at 55 °C.

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George R. Golding et al. 60

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CMRSA3 (ST239/241) CMRSA6 (ST239/241) USA700 (ST72) CMRSA7 /USA400 (ST1) CMRSA2 /USA100/USA800 (ST5) CMRSA10 /USA300 (ST8) CMRSA5 /USA500 (ST8) CMRSA9 (ST8) CMRSA1 /USA600 (ST45) CMRSA8 (ST22) CMRSA4 /USA200 (ST36) USA1100 (ST30) USA1000 (ST59)

Fig. 1 PFGE dendrogram of representative MRSA epidemic types in Canada. Dendrograms were derived from the unweighted pair group method using arithmetic averages (UPGMA) and based on Dice coefficients. Band position tolerance and optimization were set at 1.00 %. Canadian and USA PFGE epidemic type nomenclature provided with common multilocus sequence types in parenthesis

4. For data normalization, the standard should be included on both outside lanes and at least every fifth lane of the gel for each run. 5. A PFGE casting plate washed in 70 % ethanol or a sterile disposable Petri plate can be used as a cutting surface. 6. To standardize the amounts of DNA loaded on the gel, it is important to cut the same size plugs for each sample. 7. Ensure all the plug slices are completely submerged in the buffer. 8. Run times are suggested. Electrophoresis should continue until standards are within 1 in. of the bottom of the gel. 9. Pressure can be applied to the bottom black gel tray with a sterile pipette tip to push the bubbles out from underneath the tray. 10. Ethidium bromide staining solution (EtBr) and water used for destaining of gels should be changed weekly. References 1. Golding GR, Levett PN, McDonald RR et al (2011) High rates of Staphylococcus aureus USA400 infection, Northern Canada. Emerg Infect Dis 17:722–725 2. Simor AE, Gilbert NL, Gravel D et al (2010) Methicillin-resistant Staphylococcus aureus colonization or infection in Canada: National surveillance and changing epidemiology, 1995–2007. Infect Control Hosp Epidemiol 31:348–356

3. Golding GR, Bryden L, Levett PN et al (2010) Livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 in humans, Canada. Emerg Infect Dis 16:587–594 4. Voss A, Loeffen F, Bakker J et al (2005) Methicillin-resistant Staphylococcus aureus in pig farming. Emerg Infect Dis 11:1965–1966 5. Golding GR, Campbell JL, Spreitzer D et al (2008) A preliminary guideline for the assignment

Pulsed-Field Gel Electrophoresis of Staphylococcus aureus

6.

7.

8.

9.

10.

11.

of methicillin-resistant Staphylococcus aureus to a Canadian pulsed-field gel electrophoresis epidemic type using spa typing. Can J Infect Dis Med Microbiol 19:273–281 Harmsen D, Claus H, Witte W et al (2003) Typing of methicillin-resistant Staphylococcus aureus in a university setting by using a novel software for spa repeat determination and database management. J Clin Microbiol 41: 5442–5448 Enright MC, Day N, Davies CE et al (2000) Multilocus sequence typing for characterization of methicillin-resistant and methicillinsusceptible clones of Staphylococcus aureus. J Clin Microbiol 38:1008–1015 Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37:67–75 Carle GR, Olson MV (1984) Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res 12:5647–5664 Gardiner K, Laas W, Patterson D (1986) Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis. Somat Cell Mol Genet 12:185–195 Carle GF, Frank M, Olson MV (1986) Electrophoretic separations of large DNA

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13.

14.

15.

16.

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molecules by periodic inversion of the electric field. Science 232:65–68 Chu G, Vollrath D, Davis RW (1986) Separation of large DNA molecules by contourclamped homogeneous electric fields. Science 234:1582–1585 Mulvey MR, Chui L, Ismail J et al (2001) Development of a Canadian standardized protocol for subtyping methicillin-resistant Staphylococcus aureus using pulsed-field gel electrophoresis. J Clin Microbiol 39:3481–3485 McDougal LK, Steward CD, Killgore GE et al (2003) Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 41:5113–5120 Murchan S, Kaufmann ME, Deplano A et al (2003) Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol 41:1574–1585 Tenover FC, Arbeit RD, Goering RV et al (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239

Chapter 9 The Use of Pulsed-Field Gel Electrophoresis for Genotyping of Clostridium difficile Wondwossen A. Gebreyes and Pamela R.F. Adkins Abstract Genotyping approaches are important for tracking infectious agents and can be used for various purposes. Pulsed-Field Gel Electrophoresis (PFGE) is among the highly discriminatory genotyping approaches commonly used for characterizing Clostridium difficile. Other genotyping methods used for C. difficile include Ribotyping, Restriction Endonuclease Assay (REA), Multilocus Variable Number Tandem Repeats (VNTR) Assay, and others. PFGE has a high discriminatory power, high reproducibility, and typeability. We utilized PFGE for typing C. difficile isolates of porcine and human origin. We used a macrorestriction fragment analysis of an intact genomic DNA using SmaI, a rare cutting restriction endonuclease. Using a Contour-Clamped Homogeneous Electric Field (CHEF) system with running conditions of 120° angle; initial switch time of 5 s; final switch time of 40 s with a run time of 18 h in a low-melting temperature agarose (Seakem Gold); and 0.5× TBE circulated in the CHEF system at 6 V/cm [CDC (2014) Pulsenet. http://www.cdc.gov/pulsenet/index.html. Accessed 22 Aug 2014] supported by 14 °C cooling module, we were able to separate very large DNA fragments (up to 2 Mb). Key words Clostridium difficile, Genotyping, PFGE, Subtyping techniques

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Introduction Genotyping approaches overall have been used for subtyping bacterial strains with a goal of achieving a higher resolution than phenotyping approaches could offer. Usually, genotyping methods have a better discriminatory power than most phenotyping methods. Genotyping applications include identification of the source of disease outbreaks, establishing surveillance databases, and differentiating wild-type strains from vaccine strains and other molecular epidemiology purposes. Pulsed-Field Gel Electrophoresis (PFGE) is the most commonly used and often regarded as a “gold standard” genotyping method used for fingerprinting bacterial pathogens. This method has been used since 1984 when it was first used to genotype Saccharomyces cerevisiae [2].

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_9, © Springer Science+Business Media New York 2015

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One of the unique features of PFGE is, unlike most genotyping methods, it does not require extraction of DNA as its initial steps. Instead, the initial stage is embedding freshly isolated colonies in agarose blocks. This has several advantages; first, it prevents contamination of cells as well as DNA by foreign materials. Second, PFGE does not have an amplification step, unlike most genotyping methods. The absence of amplification avoids the potential nonspecific amplification that can significantly affect the reproducibility of the method. The third advantage of PFGE is the fact that the scientific principles are straightforward. It is mainly based on restriction fragmentation. It does not involve additional molecular methods such as blotting, ligation, amplification, or sequencing, thus providing simplicity. The key challenge of PFGE is the need for a more sophisticated electrophoresis chamber to be able to separate large DNA fragments, up to 2 Mb, unlike other regular horizontal gel electrophoresis systems. Currently, the Contour-Clamped Homogeneous Electric Field (CHEF) is the system utilized by the majority of the laboratories that conduct PFGE genotyping. The need for a CHEF system in the laboratory adds to the cost of setting up PFGE and thus could be a disadvantage for resource-limited laboratories, mainly in developing nations. Besides the CHEF system, other electrophoresis systems have previously been used and considered for separating large DNA fragments. These include the Rotating Gel Electrophoresis (RGE) [3], Field Inversion Gel Electrophoresis (FIGE) [4], and Transverse Alternating-Field Electrophoresis (TAFE) [5]. Despite the various attempts and methods developed to separate macrorestriction fragments, the CHEF system remains the preferred choice. PFGE has been considered as a gold standard to genotype various bacterial pathogens, mainly foodborne pathogens. The Pulsenet global database established by the U.S. Center for Disease Control and Prevention in 1996 uses PFGE as the core genotyping method for foodborne outbreak investigation (http://www.cdc. gov/pulsenet/). The Pulsenet system has recently been expanded, and the Pulsenet International aims to achieve the same goals worldwide. The Pulsenet system does not include C. difficile in its database. However, PFGE has been used in various research and clinical laboratories for subtyping C. difficile and for tracking outbreaks. C. botulinum, an important foodborne pathogen, is the only Clostridium species currently included in the Pulsenet database system. PFGE has been used as one of the key genotyping methods for subtyping C. difficile. Several studies that compared PFGE with other genotyping methods also found it to be highly discriminatory as well as a reproducible approach [6–8]. The use of PFGE in C. difficile has also enabled tracking and identification of numerous outbreaks as well as epidemic dissemination of strains. One recent example is the multistate outbreak of North American Pulsed-Field Type 1 (NAP-1), a hypervirulent strain of C. difficile

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that caused numerous nosocomial and community-acquired infections [9]. Various strains of community-acquired C. difficile in the United States have also been subtyped effectively using the PFGE method [10]. In this chapter, we outline the details of the materials and methods used for conducting effective PFGE genotyping in C. difficile.

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Materials Prepare all solutions using ultrapure water and analytical grade reagents. Buffer solutions can be made several days in advance and stored at 18–22 ºC unless otherwise noted.

2.1 C. difficile Subculture

1. Tryptic Soy Agar (TSA) plates with 5 % sheep blood and anaerobic incubation system (see Note 1).

2.2 Preparing Agarose Plugs and C. difficile Cell Lysis

1. Cell Suspension Buffer: 100 mM Tris, 100 mM EDTA (pH 8.0). Add 100 mL of 1 M Tris (pH 8), 200 mL of 0.5 M EDTA (pH 8.0), and dilute to 1,000 mL with sterile ultrapure water. 2. Tris EDTA (TE) Buffer: Add 10 mL of 1 M Tris (pH 8), 2 mL of 0.5 M EDTA (pH 8.0), and dilute to 1,000 mL with sterile ultrapure water. 3. SeaKem Gold agarose (1.8 %): Weigh 0.36 g SeaKem Gold agarose in 20 mL TE buffer (see Note 2). 4. Gram Positive Lysis Buffer: 6 mM Tris, 1 M NaCl, 100 mM EDTA, 0.5 % Brij-58, 0.2 % sodium deoxycholate, and 0.5 % sodium laurylsacrosine. Prepare by adding 0.6 mL of 1 M Tris–HCl (pH 8.0), 20 mL of 5 M NaCl, 20 mL of 0.5 M EDTA (pH 8.0), 500 mg of Brij-58, 0.2 g Deoxycholate, 0.5 g sodium laurylsacrosine, and dilute to 100 mL with sterile ultrapure water (see Note 3). 5. RNAse (1 mg/ml): Boiled RNAse is often used to make sure it is not contaminated with DNAse and thus no shearing of DNA will occur. 6. Lysozyme (lyophilized powder): keep on ice when in use and return the container to the freezer as soon as possible.

2.3 Digestion of Protein and Lipid Components

1. Proteinase K enzyme-powder form.

2.4 Nucleic Acid Digestion and Electrophoresis Separation

1. Thiourea (40 mg/mL).

2. Sodium dodecyl Sulfate (SDS). 3. EDTA, 0.5 M, pH 8.0.

2. SeaKem Gold agarose (1 %). Weigh 1.5 g SeaKem Gold agarose and add it to 150 mL Tris Borate EDTA (TBE) buffer solution, microwave and mix to ensure a uniform solution. Put it into the 50–55 °C water bath to equilibrate.

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3. 0.5× TBE. This buffer is usually purchased at a 10× concentration. A 0.5× concentration can be made by diluting 20-fold (adding 100 mL of the 10× solution and diluting to a volume of 2,000 mL with ultrapure water). 4. Restriction enzymes: SmaI and XbaI. To prepare the SmaI master mix (quantities given are per plug), add 177.5 μL of ultrapure water, 20 μL of 10× restriction buffer, and 2.5 μL of Sma1 enzyme (50 U/plug). This makes a total of 200 μL/ sample. This should be incubated at 25 °C for 4 h. Xba1 is used for the reference marker, Salmonella BAA-664. Prepare the XbaI master mix as follows (quantities given are per plug): add 175 μL of ultrapure water, 20 μL of 10× restriction buffer, and 5 μL of Xba1 enzyme (100 U/plug). This is incubated at 37 °C for 2 h. 5. Global Standard reference marker Salmonella BAA-664. 2.5 Staining and Analysis

1. Ethidium Bromide.

2.6 Equipment Required

1. Spectrophotometer.

2. Deionized water.

2. Contour-Clamped Homogeneous Electric Field (CHEF) system. 3. Water bath. 4. Plug molds.

3

Methods

3.1 Preparing the Culture (Day 1)

1. Inoculate the cultures onto TSA agar plates with 5 % sheep blood and incubate anaerobically for 48 h at 35 °C. Pick a single colony and proceed with the following steps.

3.2 Preparing the Plugs and Lysing the Organism (Day 2)

1. Make a 1.8 % agar for plug and put it into the 60–65 °C water bath. 2. Using a sterile swab, add small amounts of culture to 2 mL of Cell Suspension buffer until an absorbance (optical density) of 1.3–1.4 is reached. Optical density is measured using spectrophotometer with a 610 nm wavelength. 3. Centrifuge the cells at 8,000 × g for 5 min and remove the supernatant. 4. Resuspend the cells in 300 μL of Gram positive lysis buffer. 5. After resuspension, vortex the cells to emulsify them and put the suspension into a 37 °C water bath for 5 min. 6. Put the plug mold together and put it on ice.

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7. Add 350 μL of the 1.8 % agar (made in step 1) to the resuspended pellet, mix, and dispense it into the plug molds, adding approximately 50 μL of the solution to each plug mold. 8. Place the plug molds on ice for 15 min. 9. Make enough of the following solution to have 3 mL per plug plus extra: • (X = 3 [number of plugs currently making + 1]). • X mL Gram Positive Lysis Buffer. • (X × 20) μL 20 mg/mL solution RNase (1 mg/mL). • (X × 5) mg Lysozyme (5 mg/mL). Transfer the plugs into 3 mL of Gram positive lysis buffer with RNAse and lysozyme, and incubate in a 35 °C water bath overnight. 3.3 Treating with Proteinase K (Day 3)

1. Make enough buffer with Proteinase K and SDS for 3 mL per plug, plus extra: ●

(X = 3 [number of plugs currently making + 1]).

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X mL EDTA, 0.5 M, pH 8.0.

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(X × 10) mg Sodium dodecyl sulfate.

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X mg Proteinase K (powder).

Equilibrate in the 50–55 °C degree water bath. 2. Pour off the Gram Positive Lysis buffer with RNAse/Lysozyme and add 3 mL buffer with Proteinase K and SDS. Put this into the 52 °C degree water bath for at least 2 h or overnight. 3.4 Digestion and Running (Day 4)

1. Pour off the buffer with Proteinase K and SDS and wash four times with 4 mL of TE buffer, shaking or rotating approximately every 5 min for 30 s (this can also be done with a shaking water bath, for 30 min). Blot each time between washes. After the last wash, cover with 4 mL of TE buffer and refrigerate until ready to use or proceed to next step. These can be kept refrigerated for several weeks. 2. Make a SmaI master mix times how many plugs you are running plus a little extra. Distribute into labeled microcentrifuge tubes. Additional Step—BSA can be added to minimize the incidence of incomplete restriction. If it is decided to add this, subtract from the total water volume. 3. Cut three or four slices of the plug. Use a scalpel blade and make 2.0–2.5 mm wide slices. Add the small plug slices to the Buffer IV/SmaI/BSA mixture and incubate at 25 °C for 4 h. 4. Also, set up a sufficient number of Global Standard Salmonella BAA-664 plugs to run one every five to six plugs on the comb using XbaI enzyme (see Note 4). 5. Make 2,000 mL of 0.5× TBE buffer solution (100 mL TBE buffer + 1,900 R/O water and mix well) (see Note 5).

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6. Make 1 % agar for the gel. 7. Put the CHEF system gel form together and make sure it is level. 8. Load the comb by removing the plug slices from the tubes and remove excess buffer with a Kim wipe. Placing the plug slices on the end of each tooth of the comb. Let the plug slices air dry for 15 min on the comb. 9. Position the comb in the gel form with the plus slices correctly aligned on the bottom of the teeth and the lower edge of the plug slice against the black platform of the gel form. 10. Pour the gel slowly from the corner into the mold and let it solidify for 30 min. 11. Make sure the chamber is level and place the black gel frame in the chamber. 12. Put the remainder of the TBE buffer into the chamber and turn the cooling module along with the pump on. Make sure the temperature reaches 14 °C before running. 13. Set chamber parameters by pressing “Two State” (planning to check out some different run times): ●

Gradient = 6.

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Run time = 18 h.

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Included angle = 120.

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Int. Sw. Tm. = 5.0 s.

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Fin. Sw. Tm. = 40 s.

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Ramping Factor = Linear (just press enter).

14 Remove the comb. The wells do not need to be closed with agarose after removing the comb. Put the gel with the black backing into the chamber and press “Start Run” and let it run for 18 h. 3.5 Staining and Analyzing (Day 5)

1. Make a solution of 25 μL Ethidium Bromide and 500 mL R/O water. Add the gel and let it stain for 15 min and agitate it for 30 s in a gentle speed every 5 min. 2. Pour off the staining solution. 3. Add 1,000 mL of R/O water to the gel and rotate for 45–60 min to destain. 4. Take a photograph using a gel documentation system to digitize images and analyze using Bionumerics or related software.

4

Notes 1. The use of an anaerobic chamber is preferred for the initial isolation of C. difficile. However, for subcultures, use of anaerobic sachets in a closed container works well.

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2. A low melting point ultrapure agarose, SeaKem Gold, works very well for PFGE and is therefore highly recommended. We see minimal plug breakage with this agarose. 3. It is recommended to make the cell lysis buffer prior to starting the protocol and to store it in a 4 °C refrigerator. 4. Salmonella BAA-664 is used for a control and band size marker. The plugs for this sample can be made using a Salmonella protocol; we suggest using the PulseNet protocol (http://www. cdc.gov/pulsenet/index.html). We make a large batch of these plugs at one time and store them in a 4 °C refrigerator and use as needed. These can be stored at 4 °C for several weeks. 5. Optional Step if smearing is a problem (strain dependent): Add 760 μL thiourea (40 mg/mL—this should end up with a 200 μM solution) to the TBE buffer solution.

Acknowledgements The authors would like to thank collaborators at the CDC anaerobe laboratory, including Brandi Limbago, for assistance with this protocol. References 1. CDC (2014) Pulsenet. http://www.cdc.gov/ pulsenet/index.html. Accessed 22 Aug 2014 2. Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37(1):67–75 3. Serwer P, Hayes SJ (1989) A new mode of rotating gel electrophoresis for fractionating linear and circular duplex DNA: the effects of electrophoresis during the gel’s rotation. Appl Theor Electrophor 1(2):95–98 4. Carle GF, Carle GF (1992) Field-inversion gel electrophoresis. Methods Mol Biol 12:3–18 5. Gardiner K (1992) Transverse alternating-field electrophoresis. Methods Mol Biol 12:51–61 6. Northey G, Gal M, Rahmati A, Brazier JS (2005) Subtyping of Clostridium difficile PCR ribotype 001 by REP-PCR and PFGE. J Med Microbiol 54:543–547 7. Killgore G, Thompson A, Johnson S, Brazier J, Kuijper E, Pepin J, Frost EH, Savelkoul P, Nicholson B, van den Berg RJ, Kato H, Sambol SP, Zukowski W, Woods C, Limbago B, Gerding DN, McDonald LC (2008) Comparison of seven techniques for typing international epidemic

strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing. J Clin Microbiol 46(2):431–437 8. Tenover FC, Akerlund T, Gerding DN, Goering RV, Boström T, Jonsson AM, Wong E, Wortman AT, Persing DH (2011) Comparison of strain typing results for Clostridium difficile isolates from North America. J Clin Microbiol 49(5):1831–1837 9. McDonald LC, Killgore GE, Thompson A, Owens RC Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN (2005) An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353(23):2433–2441 10. Limbago BM, Long CM, Thompson AD, Killgore GE, Hannett GE, Havill NL, Mickelson S, Lathrop S, Jones TF, Park MM, Harriman KH, Gould LH, McDonald LC, Angulo FJ (2009) Clostridium difficile strains from community-associated infections. J Clin Microbiol 47(9):3004–3007

Chapter 10 Molecular Subtyping of Clostridium botulinum by Pulsed-Field Gel Electrophoresis Carolina Lúquez, Lavin A. Joseph, and Susan E. Maslanka Abstract Pulsed-field gel electrophoresis (PFGE) has been extensively used to estimate the genetic diversity of Clostridium botulinum. In addition, PFGE is the standard method for investigating foodborne outbreaks associated with various enteric pathogens, including C. botulinum. PFGE can be used to exclude a suspected but not confirmed food source when the patterns of the food and clinical isolates are different. Indistinguishable PFGE patterns may also be useful for linking isolates between patients or to a food source, but results must be interpreted within an epidemiological context to ensure isolates are truly related. Here, we describe a standardized laboratory protocol for molecular subtyping of C. botulinum by PFGE. Key words Pulsed-field gel electrophoresis, Clostridium botulinum, Botulinum toxin-producing clostridia, Subtyping

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Introduction Clostridium botulinum and some strains of C. butyricum and C. baratii produce botulinum neurotoxin (BoNT) which causes botulism, a rare but potentially fatal paralytic disease. There are seven confirmed serotypes of BoNT (A through G), defined by neutralization of toxicity by serotype-specific antibodies. Four naturally occurring types of botulism have been described. Foodborne botulism is always caused by ingestion of toxin contaminated with bot toxin. That is the definition of foodbone botulism. Wound botulism is caused by BoNT-producing clostridia that grow and produce toxin in an infected wound. Infant botulism and adult colonization are caused by BoNT-producing clostridia that grow and produce toxin in the intestinal tract of infants or adults, respectively. While detection of BoNT in clinical specimens, food sources, and cultures is the primary focus for laboratory investigations of botulism, molecular subtyping may help to improve the detection of outbreaks and aid in confirming or eliminating suspect food vehicles.

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_10, © Springer Science+Business Media New York 2015

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Pulsed-field gel electrophoresis (PFGE) has been extensively used to assess the genetic diversity of BoNT-producing clostridia. In a study reported by Hyytiä et al., C. botulinum type E strains obtained from various fish products showed a high diversity by PFGE [1]. Likewise, Nevas et al. reported a high diversity of proteolytic C. botulinum strains isolated from several sources in the United States and Europe [2]. Similarly, Umeda et al. showed that several C. botulinum type B strains isolated from infant botulism cases in Japan were distinguishable by PFGE [3]. PFGE was also shown to be effective in linking clinical and food C. botulinum isolates during foodborne outbreaks in Canada [4]. In a recent study, C. botulinum type A strains (subtypes A2 and A3) were distinguishable by PFGE, even when the neurotoxin gene sequences were nearly identical [5]. PulseNet, a molecular surveillance network, was established in the United States in 1996 for the laboratory detection and investigation of foodborne outbreaks [6] and has expanded to include international networks in Canada, Europe, Asia, and Latin America [7]. The success of this network is attributed to the use of rigorously standardized protocols, trained and competency assessed staff, and a database of patterns accessible by network participants. PFGE is considered a “gold standard” method, both in the United States and internationally, for investigating foodborne outbreaks resulting from contamination of the food supply by bacteria such as Shiga toxin-producing Escherichia coli, Salmonella, and Listeria monocytogenes. With some exceptions, PFGE can be used to exclude a suspected food source when the patterns of the food and clinical isolates are distinguishable. Additionally, isolates with indistinguishable PFGE patterns but from different samples may be considered related as long as an epidemiological link is known (Fig. 1). The Centers for Disease Control and Prevention (CDC) and the Virginia Department of Health, Division of Laboratory Services (supported by the Association of Public Health Laboratories), codeveloped a standardized PFGE protocol for subtyping C. botulinum using SmaI as the primary restriction enzyme and XhoI as the secondary enzyme. The procedure was approved by PulseNet in 2010. In 2012, the US PulseNet database was expanded to include C. botulinum patterns and currently contains over 150 unique SmaI patterns and 130 unique XhoI patterns representing over 400 isolates.

2

Materials

2.1 Preparation of Plugs

1. Anaerobic blood agar plates containing 5 % sheep blood, L-cystine, hemin, and vitamin K1 (CDC formulation). 2. Cell suspension buffer: 100 mM Tris–HCl, 100 mM EDTA. Dilute 100 mL of 1 M Tris–HCl, pH 8 and 200 mL

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Fig. 1 Example of C. botulinum PFGE patterns. Plugs in lanes 2, 3, 4, and 9 were restricted with SmaI. Plugs in lanes 6, 7, and 8 were restricted with XhoI. Lanes 1, 5, and 10 represent the standard Salmonella ser Braenderup H9812 restricted with XbaI

of 0.5 M EDTA, pH 8 to a volume of 1,000 mL with sterile ultrapure water. Sterilize and store at 18–22 °C (see Note 1). 3. 12 mm × 75 mm Falcon tubes or equivalent. 4. MicroScan® turbidity meter or equivalent turbidity meter. 5. Microcentrifuge. 6. Egg yolk agar plates: 7.5 % McClung Toabe agar base, 0.5 % yeast extract, 10 % egg yolk. 7. Stationary and shaking water baths. 8. Tris–EDTA buffer (TE buffer). 10 mM Tris–HCl, 0.1 mM EDTA, pH 8. Dilute 10 mL of 1 M Tris, pH 8 and 2 mL of 0.5 M EDTA to a volume of 1,000 mL with sterile ultrapure water. Sterilize and store at 18–22 °C (see Note 1). 9. Cell lysis buffer: 12 mM Tris–HCl, 2 M NaCl, 200 mM EDTA, 1 % Brij 58, 0.4 % deoxycholate, 5 % N-lauroylsarcosine (Sarkosyl). Dilute 1.2 mL of 1 M Tris–HCl, pH 8, 40 mL of 5 M NaCl, 40 mL of 0.5 M EDTA, 1 g of Brij 58, 0.4 g of deoxycholate, and 5 g of Sarkosyl to a volume of 100 mL with sterile ultrapure water. Heat the solution on a hot plate while stirring until all of the solids are dissolved. Sterilize and store at 18–22 °C (see Note 1). 10. PIV buffer. 10 mM Tris–HCl, 1 M NaCl. Dilute 5 mL of 1 M Tris–HCl, pH 8 and 100 mL of 5 M NaCl to a volume of

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500 mL with sterile ultrapure water. Sterilize and store at 18–22 °C (see Note 1). 11. Proteinase K, 20 mg/ml. 12. Lysozyme stock solution: 20 mg/mL lysozyme in TE buffer. Weigh 1 g of lysozyme. Add to 50 mL of TE buffer and swirl to mix. This stock solution can be aliquoted and stored at −20 °C. 13. Mutanolysin stock solution: 5 U/μL mutanolysin in TE buffer. Add 1 mL of TE buffer to a vial of lyophilized mutanolysin; swirl to mix. This stock solution can be aliquoted and stored at −20 °C. 14. 1.2 % SeaKem Gold agarose in TE buffer (see Note 2). Weigh 0.12 g of SeaKem® Gold agarose into a 250 mL screw-cap flask. Add 10 mL of TE buffer; swirl gently to disperse the agarose. Loosen the cap and microwave for 30 s. Mix gently and repeat for 10 s intervals until the agarose is completely dissolved. Place the flask in a 55 °C water bath until ready to use. 15. Reusable PFGE plug moulds. 16. EDTA–Sarkosyl buffer (ES buffer): 0.1 % Sarkosyl in 0.5 M EDTA. Mix 0.5 g of Sarkosyl in 500 mL of 0.5 M EDTA, pH 8. Heat the solution on a hot plate while stirring until all of the solids are dissolved. Sterilize and store at 18–22 °C (see Note 1). 17. Single-edge razor blades. 18. 50 mL polypropylene screw-cap tubes. 19. Screen caps. 2.2 Restriction Digestion of DNA in Agarose Plugs

1. 1× Restriction enzyme buffers. Prepare the 1× buffers immediately prior to use; make enough for each plug slice plus one additional aliquot. Dilute 20 μL of 10× SmaI restriction buffer (CutSmart buffer) in 180 μL of sterile ultrapure water. Dilute 20 μL of 10× XhoI restriction buffer (CutSmart buffer) and 1 μL of 20 mg/mL bovine serum albumin (BSA) in 179 μL sterile ultrapure water. 2. Single-edge razor blades. 3. Stationary water baths. 4. Restriction enzyme master mix. Prepare the master mix immediately prior to use. Keep the vial of restriction enzyme on ice, in an insulated storage box, or equivalent at all times. Prepare enough master mix for each plug slice plus one additional aliquot. Dilute 20 μL of 10× SmaI restriction buffer and 2.5 μL of SmaI enzyme (50 U/sample) in 177.5 μL sterile ultrapure water. Dilute 20 μL of 10× XhoI restriction buffer, 1 μL of 20 mg/mL BSA, and 5 μL of XhoI enzyme (100 U/sample) in 174 μL sterile ultrapure water (see Note 3).

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1. 0.5× Tris–borate–EDTA buffer (TBE). Dilute 125 mL of 10× TBE stock in 2,375 mL sterile ultrapure water. This is sufficient buffer for both the gel preparation and electrophoresis running buffer. 2. 1 % SeaKem Gold agarose in 0.5× TBE. Weigh 1 g of agarose into a 500 mL screw-cap flask. Add 100 mL of 0.5× TBE for a 14-cm-wide gel form (10 wells). Alternatively, weigh 1.5 g of agarose and add 150 mL of 0.5× TBE for a 21-cm-wide gel form (15 wells). Swirl gently to disperse the agarose. Loosen the cap of the flask and microwave the flask for 60 s; mix gently and repeat for 15 s intervals until the agarose is completely dissolved. Place the flask in a 55 °C water bath until ready to use. 3. Pulsed-field gel electrophoresis casting stand and 10-well or 15-well comb. 4. Thiourea (10 mg/mL). Weigh 1 g of thiourea. Dissolve thiourea in 100 mL of sterile ultrapure water in a sterile screw-cap glass bottle or flask. Cover the flask with aluminum foil and store in the dark (see Note 4). 5. CHEF Mapper or equivalent equipment. 6. Ethidium bromide solution. Dilute 40 μL of ethidium bromide stock solution (10 mg/mL) with 400 mL of sterile ultrapure water. This volume is for a staining container that is approximately 14 cm × 24 cm; a larger container may require a larger amount of staining solution. Store diluted ethidium bromide solution in the dark. Alternatively, gels can be stained with GelStar (see Note 5). 7. Gel Doc™ XR+ imaging system or equivalent equipment.

3

Methods Laboratory workers may be at risk of botulism by exposure to BoNT through accidental ingestion, inhalation, or contact with eyes, mucous membranes, or broken skin. Biosafety Level 2 practices, containment equipment, and facilities are recommended. Solutions of sodium hypochlorite (0.1 %) or sodium hydroxide (0.1 N) are recommended for decontaminating work surfaces and spills [8]. All supplies that come in contact with cultures, cell suspensions, or plugs should be treated as contaminated materials and should be discarded or disinfected in accordance to your institution’s biosafety guidelines. Reusable supplies should be soaked in 10 % bleach for at least 1 h before they are washed.

3.1 Preparation of Plugs

1. Streak an isolated colony of C. botulinum onto an anaerobic blood agar plate for isolation. Incubate anaerobically at 35 °C overnight.

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2. Transfer 2 mL of cell suspension buffer to the appropriate number of 12 mm × 75 mm Falcon tubes or equivalent. Use a sterile polyester fiber or cotton swab that has been moistened with sterile cell suspension buffer to remove some of the growth from the anaerobic blood agar plates. Suspend the cells in cell suspension buffer by spinning the swab gently. 3. Adjust the concentration of cell suspensions to 0.18–0.20 using a turbidity meter (measured in the 12 mm × 75 mm Falcon tubes) by diluting with sterile cell suspension buffer or by adding additional cells. 4. Transfer 1 mL of the cell suspensions into sterile microcentrifuge tubes and centrifuge for 5 min at 1,400 × g. 5. Remove the supernatants and discard into a waste container containing 10 % bleach. Resuspend the cells in 1 mL of cell suspension buffer and centrifuge for 5 min at 1,400 × g. 6. Remove the supernatants and discard into a waste container containing bleach. Resuspend cells in 500 μL of cell suspension buffer. 7. Streak 250 μL of these washed cells onto egg yolk agar plates for confluent growth. Incubate the plates anaerobically at 35 °C overnight. 8. Pre-warm sterile ultrapure water, TE buffer, and cell lysis buffer in a water bath at 55 °C. 9. Transfer 1.5–2 mL of PIV buffer to small tubes (12 mm × 75 mm Falcon tubes or equivalent). Use a sterile polyester fiber or cotton swab that has been moistened with sterile PIV buffer to remove some of the growth from the egg yolk agar plates; suspend the cells in PIV buffer by spinning the swab gently so that the cells will be evenly dispersed. 10. Adjust the concentrations of the cell suspensions to 0.68–0.72 as measured on MicroScan Turbidity Meter by diluting with sterile PIV buffer or by adding additional cells (see Note 6). 11. Transfer 1 mL of the cell suspensions into microcentrifuge tubes and centrifuge for 5 min at1,400 × g. 12. Remove the supernatants and discard them into a waste container containing bleach. Resuspend the cells in 283 μL of cell lysis buffer μL (see Note 7). 13. Add 80 μL of lysozyme (20 mg/mL) and incubate in a water bath at 55 °C for 20 min. 14. Add 4 μL of mutanolysin (5 U/μL) and 33 μL of proteinase K (see Note 7). Incubate in a water bath at 37 °C for 10 min. 15. Add 400 μL of melted 1.2 % SeaKem Gold agarose to the cell suspensions; mix by gently pipetting the mixture up and down a few times. Over-pipetting can cause DNA shearing.

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16. Immediately, dispense the agarose into wells of reusable plug mould. Do not allow air bubbles to form. Two plugs of each sample can be made from these cell suspensions. 17. Allow the plugs to solidify at 18–22 °C for about 15 min. 18. Add 5 mL of ES buffer to 50 mL polypropylene screw-cap tubes with screened caps (one tube per sample) (see Note 8). 19. Add 35 μL of proteinase K to each tube containing ES buffer (see Note 9). 20. Trim excess agarose from the top of the plug mould with a razor blade or similar instrument. Open the reusable plug mould. Using a spatula, transfer the plugs from the mould to the tubes containing ES buffer and proteinase K. Ensure the plugs are under buffer and not on the side of tube. 21. Incubate the tubes in a water bath at 55 °C with constant agitation (70 rpm) for a minimum of 2 h (see Note 10). 22. Remove the tubes from the water bath and carefully pour off the ES buffer into a discard container with bleach. Remove all of the liquid by touching the edge of the tube on an absorbent paper towel. 23. Add 20 mL of pre-warmed sterile ultrapure water to each tube. Incubate the tubes in a water bath at 55 °C, 15 min with constant agitation (70 rpm). 24. Decant used sterile ultrapure water into a waste container containing bleach. Remove all of the liquid by touching the edge of the tube on an absorbent paper towel. 25. Repeat the wash step with pre-warmed water. 26. Decant used sterile ultrapure water into a waste container containing bleach. Remove all of the liquid by touching the edge of tube on an absorbent paper towel. 27. Add 20 mL of TE buffer pre-warmed at 55 °C. Incubate the tubes in a water bath at 55 °C, 15 min with constant agitation (70 rpm). 28. Decant the TE buffer into a waste container containing bleach. Remove all of the liquid by touching edge of tube on an absorbent paper towel. 29. Repeat the wash step with pre-heated TE buffer five more times. 30. Decant the TE buffer and add 5 mL of TE buffer. Plugs can be stored in TE buffer at 4 °C until needed (for up to 4 months). 3.2 Restriction Digestion of DNA in Agarose Plugs

1. Add 200 μL of appropriate 1× restriction enzyme buffer to 1.5 mL microcentrifuge tubes. 2. Carefully remove the plugs from the TE buffer with a spatula, and place them on a disposable Petri dish or on glass slide.

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3. Cut a 2–2.5 mm wide slice from each plug with a razor blade and transfer to a tube containing 1× restriction enzyme buffer. Ensure the plug slice is under buffer. Replace the rest of plug into the original tube that contains TE buffer and store at 4 °C. 4. For plugs that will be digested with SmaI, incubate plug slices in a water bath at 25 °C for 10 min. For plugs that will be digested with XhoI, incubate plug slices in a water bath at 37 °C for 10 min. 5. Remove 1× restriction enzyme buffer from each tube; be careful not to damage the plug slice. 6. Add 200 μL of restriction enzyme master mix containing SmaI or XhoI (). Close the tube and mix by gently tapping. Ensure the plug slices are under the enzyme mixture. 7. Incubate plug slices with SmaI in a water bath at 25 °C for 4 h; incubate plug slices with XhoI in a water bath at 37 °C for 3 h. 3.3 Agarose Gel Casting and Electrophoresis

1. Place a gel mould on a leveling table and adjust until leveled. Place the comb holder so that the front part and teeth face the bottom of the gel frame and the comb teeth touch the gel platform. 2. Remove restricted plug slices from the water baths. 3. Remove restriction enzyme master mix and add 200 μL of 0.5× TBE. Incubate at 18–22 °C for 5 min. 4. Remove the plug slices from the tubes, put the comb on the bench top, and load the plug slices on the bottom of the comb teeth. Salmonella serotype Braenderup (strain H9812) digested with restriction enzyme XbaI is used as standard reference marker [9, 10]. For 10-well gels, place standards in lanes 1, 5, and 10. For 15-well gels, place standards in lanes 1, 5, 10, and 15. 5. Remove excess buffer and allow the plug slices to air-dry on the comb for 15 min. 6. Position the comb in the gel mould. Make sure that the lower edge of the plug slices is aligned against the platform. 7. Carefully pour melted 1 % SeaKem Gold agarose in 0.5× TBE into the gel mould. Use a pipette tip to remove bubbles or debris. Allow the gel to solidify for a minimum of 15 min at 18–22 °C. 8. Put the gel frame in the electrophoresis chamber. Fill the chamber with freshly prepared 0.5× TBE (approximately 2,200 mL). Close the cover of the unit. 9. Turn on the power supply, cooling module (set to 14 °C), and pump (set at about 70 to ensure a flow rate of 1 L/min) while

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the gel is solidifying, approximately 30 min. before the gel is to be run. 10. After the gel has solidified, gently remove the comb from the gel. 11. Unscrew and remove the end gates from the gel mould; remove excess agarose from the sides and bottom of the casting platform with a tissue. Keep the gel on the casting platform and carefully place the gel inside the gel frame in the electrophoresis chamber. 12. Add 860 μL of thiourea (10 mg/mL) to the electrophoresis chamber. Close the cover of the chamber. 13. Select the following conditions on the CHEF Mapper: Auto Algorithm 30 kb: low MW 600 kb: high MW Initial switch time: 0.5 s Final switch time: 40 s Run time: 18–19 h (see Note 11) 14. Make a note of the initial milliamp (mA) reading on the instrument. The initial mA should be between 110 and 150 mA. A reading outside of this range may indicate that the 0.5× TBE buffer was prepared improperly and the buffer should be remade. 15. When the electrophoresis run is over, turn off the equipment and remove the gel. Drain the buffer from the electrophoresis chamber. Add approximately 2,500 mL of distilled water to the electrophoresis chamber and let the pump run for at least 1 h. Drain the distilled water from the electrophoresis chamber. 16. Stain the gel with ethidium bromide in a covered container for 20–30 min, with gentle agitation (see Note 12). Alternatively, gels can be stained with GelStar (see Note 5), or other staining system. 17. If the gel was stained with ethidium bromide, destain in approximately 500 mL of reagent grade water for 60–90 min; change the water every 20 min. If too much background is observed, destain for additional 30–60 min. 18. Capture the image in a Gel Doc™ XR, or equivalent documentation system. Some C. botulinum isolates may not be typeable by the procedure described above. An alternative procedure, which includes treatment with formaldehyde, can be used to improve typeability of those strains [11].

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Notes 1. Sterilize buffers by filtering through a 0.45 μm filter unit or by autoclaving at 121 °C for 15 min. 2. SeaKem Gold agarose works well for making PFGE plugs because it provides added strength to the plugs, minimizing breakage of plugs during the lysis and washing steps. The time and temperature needed to completely dissolve the agarose may have to be determined empirically in each laboratory. 3. Several restriction enzyme vendors specifically recommend the addition of BSA to the enzyme restriction mixtures while others do not. Incomplete restriction may be minimized by adding BSA. Restriction enzymes may be provided at different concentrations; calculate the volume needed to achieve the same final concentrations as indicated in this protocol. Ensure the restriction buffer used is recommended by the vendor for the corresponding restriction enzyme. 4. Thiourea is a toxic chemical; weigh thiourea in a chemical fume hood; use gloves, eye protection, and a disposable spatula when handling this chemical. Clean up any spills, and wipe down the balance and surrounding area with a moistened towel. Discard gloves, spatula, weighing paper, etc. as hazardous waste, according to the guidelines of your institution. Recap the bottle tightly after use. 5. Dilute 40 μL of 10,000× GelStar stock solution into 400 mL of 1× TBE. Stain the gel in a covered container for 60 min with gentle agitation. Destaining is not necessary with GelStar. GelStar should be filtered through activated charcoal before discarding. 6. The optimal cell suspension concentration may need to be adjusted for satisfactory results. Cell suspensions should be at 18–22 °C when concentration is measured. 7. Proteinase K may differ in concentration from lot to lot. If concentration of proteinase K is not 20 mg/mL, calculate the volume of cell lysis buffer needed per sample so that: Cell lysis buffer + proteinase K (0.665 mg/sample) = 316 μL 8. The screen allows the pouring of the liquids from reactions and washes out of the tubes without damaging or losing the plugs. 9. Proteinase K may differ in concentration from lot to lot. If concentration of proteinase K is not 20 mg/mL, calculate the volume of proteinase K needed per sample so that its final concentration is 0.14 mg/mL. 10. Plugs usually are sufficiently stable to perform washing steps at 55 °C. However, if plugs are nicked along the edges or breaking, temperature of washing steps may be lowered to 50 °C.

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11. The electrophoresis running times recommended here are based on the equipment and reagents used at CDC. 12. Stock solutions of 10 mg/mL ethidium bromide in water are available from several commercial companies. The diluted solution can be kept in a dark bottle and reused 6–8 times. Aqueous solutions containing ethidium bromide can be filtered through activated charcoal or degraded using activated carbon destaining or “tea” bags, which effectively and safely remove ethidium bromide from solutions. Once the ethidium bromide is removed, the treated aqueous solutions can be discarded. References 1. Hyytiä E, Hielm S, Björkroth J et al (1999) Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products. Appl Environ Microbiol 65:2057–2064 2. Nevas M, Lindstrom M, Hielm S et al (2005) Diversity of proteolytic Clostridium botulinum strains, determined by a pulsed-field gel electrophoresis approach. Appl Environ Microbiol 71:1311–1317 3. Umeda K, Seto Y, Kohda T et al (2009) Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan. J Clin Microbiol 47:2720–2728 4. Leclair D, Pagotto F, Farber JM et al (2006) Comparison of DNA fingerprinting methods for use in investigation of type E botulism outbreaks in the Canadian Arctic. J Clin Microbiol 44:1635–1644 5. Lúquez C, Raphael BH, Joseph LA et al (2012) Genetic diversity among Clostridium botulinum strains harboring bont/A2 and bont/A3 genes. Appl Environ Microbiol 78:8712–8718 6. Gerner-Smidt P, Hise K, Kincaid J et al (2006) PulseNet USA: a five-year update. Foodborne Pathoq Dis 3:9–19 7. Swaminathan B, Gerner-Smidt P, Ng LK et al (2006) Building PulseNet International: an

8.

9.

10.

11.

interconnected system of laboratory networks to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging foodborne diseases. Foodborne Pathoq Dis 3:36–50 Biosafety in Microbiological and Biomedical Laboratories (BMBL), (2009) 5th edn, U.S. Department of Health and Human Services, Public health Service, Centers for Disease Control and Prevention, National Institutes of Health http://www.cdc.gov/biosafety/publications/bmbl5/ Hunter SB, Vauterin P, Lambert-Fair MA et al (2005) Establishment of a universal size standard strain for use with PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol 43:1045–1050 Ribot EM, Fair MA, Gautom R et al (2006) Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathoq Dis 3:59–67 Hielm S, Björkroth J, Hyytiä E et al (1998) Genomic analysis of Clostridium botulinum group II by pulsed-field gel electrophoresis. Appl Environ Microbiol 64:703–708

Chapter 11 Pulsed-Field Gel Electrophoresis of Yersinia pestis Tamara Revazishvili and Judith A. Johnson Abstract Yersinia pestis is a human pathogen and can cause serious disease. Biosafety level 3 (BSL3) is required when handling this microorganism and all work requires a biological safety cabinet. For pulsed-field gel electrophoresis (PFGE), dedicated BSL3 PFGE equipment or a documented procedure that ensures that all viable bacteria are inactivated is required. All plasticware and glassware that comes into contact with the cultures should be disinfected/sterilized or disposed of in a safe manner, according to the guidelines of institution. This includes decontamination of pipettes, spatulas, etc. that were in contact with the cell suspensions or plugs. Disinfection of reusable plug molds should be done before they are washed; the disposable plug molds, including the tape and the tab that was used to push the plugs out of the wells, are also contaminated and should be disinfected with 10 % bleach for at least 30 min if they will be washed and reused. Key words Yersinia pestis, Pulsed-field gel electrophoresis, DNA separations, Restriction/digestion

1

Introduction Pulsed-field gel electrophoresis (PFGE) was first described in 1984 as a tool for examining the chromosomal DNA of bacterial genomes of 2,500–5,000 kb, using rare cutting restriction enzymes, followed by separation of the fragments (10–30 fragments ranging in size from 10 to 800 kb) on a gel. Essentially, these fragments are resolved by PFGE into a pattern of distinct bands. This technique is considered the “gold standard” of the methods of molecular typing, being highly discriminatory and useful for almost any bacterial pathogens, including Yersinia pestis [1, 2]. Before PFGE, larger fragments of DNA could not be separated by gel electrophoresis as they tend to move through the agarose in a linear form, a process called reptation. Linear DNA molecules are not resolved by size as they all have the same diameter and can pass through the agarose matrix at the same speed. PFGE uses a specially designed chamber that positions the agarose gel between three sets of electrodes that form a hexagon around the gel.

Kieran Jordan and Marion Dalmasso (eds.), Pulse Field Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1301, DOI 10.1007/978-1-4939-2599-5_11, © Springer Science+Business Media New York 2015

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Fig. 1 Schematic diagram of general PFGE method

Larger pieces of DNA are separated by shifting the direction of the current frequently, keeping the DNA molecules tangled up in a ball. The cumulative effect of the directional changes moves the DNA down the length of the gel and smaller DNA “balls” can pass through smaller pores in the agarose matrix more easily than larger DNA “balls” resulting in faster overall movement through the gel. Thus the periodic alternation of the angle of the direction of the electric field resolves even very large DNA fragments [3]. PFGE has remarkable discriminatory power and reproducibility [4]; the experiment takes no longer than 2–4 days and requires relatively inexpensive PFGE equipment. A schematic diagram of general PFGE method is presented in Fig. 1 [5]. PFGE can also be used to determine size of plasmids for Y. pestis (Fig. 2) [6, 7].

2 2.1

Materials Bacterial Culture

1. BHI or LB agar plates for streaking a Y. pestis isolated colony. 2. LB medium. 3. Sterile loops.

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Fig. 2 Pulsed-field gel electrophoresis of total plasmid content on 1 % agarose gel. Lane 1, low-range PFG marker; lane 2, Y. pestis C790; lane 3, Y. pestis CO92; lane 4, Y. pestis C2614; lane 5, Y. pestis C2944; and lane 6, medium range PFG marker (7) 2.2

Equipment

1. Shaker water bath (54 °C), stationary water bath (55–60 °C), or heat block. 2. Spectrophotometer (or equivalent instrument such as a turbidity meter or a colorimeter).

2.3 Buffers and Reagents Used in Protocol for PFGE

1. TE (Tris–EDTA buffer, pH 8.0) (10 mM Tris–1 mM EDTA, pH 8.0). Mix 10 mL of 1 M Tris, pH 8.0, and 2 mL of 0.5 M EDTA, pH 8.0. Dilute to 1,000 mL with sterile Ultrapure (Reagent Grade Type 1) water (see Note 1). 2. Cell suspension buffer (100 mM Tris–100 mM EDTA, pH 8.0). Mix 10 mL of 1 M Tris, pH 8.0, and 20 mL of 0.5 M EDTA, pH 8.0. Dilute to 100 mL with sterile Ultrapure (Reagent Grade Type 1) water. 3. Cell lysis buffer (50 mM Tris; 50 mM EDTA, pH 8.0; 1 % sarcosine; 0.1 mg/mL Proteinase K). Mix 25 mL of 1 M Tris, pH 8.0; 50 mL of 0.5 M EDTA, pH 8.0; and 50 mL of 10 % N-lauroylsarcosine, sodium salt (Sarcosyl), or 5 g of Nlauroylsarcosine, sodium salt (Sarcosyl). Dilute to 500 mL

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with sterile Ultrapure (Reagent Grade Type 1) water. Add 25 μL Proteinase K (20 mg/Ml) to 5 mL of cell lysis buffer before use. The final concentration of Proteinase K in the lysis buffer should be 0.1 mg/mL. In a case where Sarcosyl powder is added directly to the other components, warm the solution to 50–60 °C for 30 min or leave at 18–25 °C to completely dissolve the Sarcosyl. 4. 10 % Sarcosyl (N-lauroylsarcosine, sodium salt). Add 10 g Sarcosyl to 90 mL of sterile water. Mix gently and warm up to 50–60 °C. 5. Proteinase K stock solution (20 mg/mL). Add 100 mg of Proteinase K powder to 5 mL of sterile water. Mix and aliquot 500 μL. Store at −20 °C. 6. 1 M Tris–HCl, pH 8.0. Dissolve 121.1 g Tris base in 650– 700 mL water. Add approximately 80 mL of 6 N HCl. Cool to 18–25 °C. Adjust the pH to 8.0 and dilute to 1,000 mL with Type 1 water. Autoclave at 121 °C for 15 min. Another procedure can be to dissolve 157.6 g of Tris base in 800 mL of Type 1 water. Cool to 18–25 °C and adjust the pH. Dilute to 1,000 mL with Type 1 water before autoclaving as previously mentioned. 7. 10 N NaOH. Dissolve 400 g NaOH in 800 mL of Type 1 water. Cool to 18–25 °C. Dilute to 1,000 mL with Type 1 water. 8. 0.5 M EDTA, pH 8.0. Add 186.1 g Na2EDTA to 800 mL of Type 1 water. Adjust the pH to 8.0 with approximately 50 mL of 10 N NaOH added slowly. Dilute to 1,000 mL with Type 1 water. Autoclave at 121 °C for 15 min. 9. 10 % sodium dodecyl sulfate (SDS). Add 10 g SDS to 90 mL of sterile Type 1 water. Mix gently and warm to 40–45 °C. 10. 10× Tris-borate EDTA buffer (TBE) (0.9 M Tris, 0.9 M boric acid, 0.02 M EDTA). Mix 108 g Tris, 55 g boric acid, and 40 mL of 0.5 M EDTA, pH 8.0. Dilute to 1,000 mL with sterile water. Autoclave at 121 °C for 15 min. 11. Ethidium bromide (10 mg/mL stock solution). 12. SeaKem Gold (SKG) agarose.

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Methods

3.1 Bacteria Growth Conditions

1. The isolate is grown at 28–30 °C for 24–48 h, in LB or BHI broth and on LB or BHI agar. If additional diagnostic tests for the presence of F1 antigen are to be done on the isolate, cultures need to be incubated at 37 °C for the F1 antigen to be expressed.

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2. Prepare 1 % agarose for PFGE plugs by weighing 0.5 g (or 0.25 g) of SKG agarose into a 250-mL screw-cap flask. Add 47 mL (or 23.5 mL) TE buffer; swirl gently to disperse the agarose. Loosen the cap and microwave for 30 s; mix gently and repeat for 10-s intervals until the agarose is completely dissolved. Place the flask in a 55–60 °C water bath or heat block for 5 min before adding SDS. Add 2.5 mL (or 1.25 mL) of preheated to 55 °C 20 % SDS and mix well. Recap the flask and return it to the 55–60 °C water bath or heat block until it is ready to use (see Note 2). 3. Label hemolysis tubes (or equivalent) with culture numbers. 3.2 Cell Suspension and Casting Plugs

1. Transfer 2 mL of CSB to the labeled tubes. 2. Use a sterile polyester fiber or cotton swab to remove some bacterial growth from agar plates. Suspend cells in CSB by rotating the swab gently so that the cells will be evenly dispersed and formation of aerosols is minimized (see Note 3). 3. Measure the optical density (OD) of the culture at 610 nm wavelength. The OD should be around 1.35 (range of 1.3– 1.4) (see Note 4).

3.3 Preparation of the Plugs

1. Label the wells of PFGE plug molds with the culture numbers. 2. Transfer 400 μL of adjusted cell suspensions to labeled 1.5-mL microcentrifuge tubes. If the cell suspensions are at 18–25 °C, agarose can be added directly without pre-warming the cell suspensions. If the cell suspensions are cold, incubate in a 37 °C water bath or heat block for a few minutes. 3. Add 20 μL of Proteinase K to each tube and mix gently with the pipette tip. 4. Add 400 μL of melted 1 % agarose to the 400 μL of cell suspension; mix gently by pipetting up and down a few times. Maintain the temperature of the melted agarose by keeping the flask in a warm water bath (55–60 °C) or heat block. Immediately dispense part of the mixture into appropriate well(s) of the disposable plug mold. Do not allow air bubbles to form. Two plugs of each sample can be made from these amounts of cell suspension and agarose. Allow the plugs to solidify at 18–25 °C for 10–15 min. They can also be placed in the refrigerator (4 °C) for 5 min. Disposable plug molds are also recommended for BSL3 work as the plug molds can be discarded easily. For disposable plug molds, use 200 μL cell suspension, 10 μL of Proteinase K, and 200 μL of agarose. Up to four plugs can be made from these amounts of cell suspension and agarose (see Notes 5 and 6).

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3.4 Cell Lysis in Agarose Plugs ( See Note 7)

1. Label 50-mL polypropylene screw-cap tubes with culture numbers. 2. Prepare the Proteinase K/cell lysis buffer. For each tube, 5 mL of cell lysis buffer and 25 μL Proteinase K are needed. Measure the correct volumes into appropriate size test tube or flask for the number of samples tested, and mix well (see Note 8). 3. Add 5 mL of Proteinase K/cell lysis buffer to each labeled 50-mL tube. 4. Trim the excess of agarose from the top of the plugs with a scalpel or razor blade. Open the reusable plug mold and transfer the plugs from the mold with a spatula to the appropriately labeled tubes. If disposable plug molds are used, remove the tape from the bottom of the mold and push out the plug(s) into the appropriately labeled tubes. Ensure the plugs are under the buffer and not stuck to the side of the tube (see Note 9). 5. Place the tubes in a rack and incubate in a 54 °C shaking water bath for 2 h with constant and vigorous agitation (175– 200 rpm). Be sure the water level in the water bath is above the level of lysis buffer in the tubes. Plugs can be lysed for longer periods of time (3–16 h), if required. 6. Preheat enough sterile Ultrapure (Reagent Grade Type 1) water to 50 °C that plugs can be washed two times with 10–15 mL water (200–250 mL for 10 tubes).

3.5 Washing Agarose Plugs

1. Preheat a shaking water bath to 50 °C. 2. Remove the tubes from the 54 °C water bath, and carefully discard the lysis buffer into an appropriate container. The plugs can be held in tubes with a screened cap (see Note 10). 3. Add 10–15 mL of sterile Ultrapure (Reagent Grade Type 1) water, which has been preheated to 50 °C, to each tube and shake the tubes vigorously in the 50 °C water bath for 10–15 min. 4. Discard the water from the plugs and repeat the wash step with preheated water (step 3) one more time. 5. Preheat enough sterile TE buffer in the 50 °C water bath so that plugs can be washed four times with 10–15 mL TE. 6. Discard the water, add 10–15 mL of preheated TE buffer, and shake the tubes vigorously in the 50 °C water bath for 10–15 min. Plug washing with TE can be done for 30–45 min and at 37 °C or at 18–25 °C. Plugs can also be refrigerated overnight in TE. 7. Discard TE buffer and repeat the wash step with preheated TE, three times. 8. Discard TE buffer and add 5–10 mL of sterile TE buffer. Store the plugs at 4 °C for up to 6 months.

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Table 1 Pre-restriction incubation calculation for enzyme XbaI [10] Reagent

μL/plug slice

μL/10 plug slices

Sterile water

180

1,800

20

200

200

2,000

Buffer H Total volume

Table 2 Pre-restriction incubation calculation for enzyme AscI Reagent

μL/plug slice

μL/10 plug slices

Sterile water

180

1,800

20

200

200

2,000

Buffer 4 Total volume

3.6 Restriction/ Digestion of DNA in Agarose Plugs with Restriction Enzymes AscI and XbaI ( See Note 11)

1. Label 1.5-mL microcentrifuge tubes with Y. pestis culture numbers. 2. Dilute 10× Buffer 4 or 10× Buffer H ten times with sterile Ultrapure (Reagent Grade Type 1) water according to Tables 1 and 2. 3. Add 200 μL of diluted Buffer 4 (1×) for enzyme AscI (Fig. 3) [8] or Buffer H (1×) for enzyme XbaI (Fig. 4) [8] to labeled 1.5-mL microcentrifuge tubes. 4. Carefully remove a plug from the TE with a spatula, and place it in a sterile disposable Petri dish or on a large glass slide. 5. Cut a 2.0–2.5-mm-wide slice with a single-edge razor blade (or scalpel, cover slip, etc.) and transfer the slice to the tube containing the diluted Buffer 4 or Buffer H. Be sure the plug slice is under buffer. Replace the rest of the plug in its original tube containing TE buffer. Store at 4 °C (see Note 12). 6. Incubate the plug slices in a 37 °C water bath for 5–10 min. 7. After incubation, remove the buffer with a micropipette fitted with a 200 μL tip all the way to the bottom of the tube and aspirate the buffer. Be careful not to cut the plug slice with the pipette tip. 8. Dilute the 10× Buffer 4 or 10× Buffer H ten times with sterile Ultrapure (Reagent Grade Type 1) water according to Tables 3 and 4. Add the restriction enzyme according to Tables 3 and 4. Mix in the same tube that was used for the diluted buffer (see Note 13).

Fig. 3 Representative PFGE patterns of AscI digested DNA of Y. pestis strains. Lanes 1 and 15, λ ladder PFG marker; lanes 2 and 14, MidRange PFG marker; lanes 3–6, PFGE-type P1AscI; lane 7, PFGE-type P5AscI; lane 8, PFGE-type P6AscI; lane 9, PFGE-type P7AscI; lane 10, PFGE-type P2AscI; lane 11, PFGE-type P3AscI; lane 12, PFGE-type P4AscI; and lane 13, PFGE-type P8AscI (8)

Fig. 4 Representative PFGE patterns of XbaI-digested DNA of Y. pestis strains. Lanes 1 and 15, λ ladder PFG marker; lanes 2 and 14, MidRange PFG marker; lanes 3–5 and 10–13, PFGE-type P1XbaI; lane 6, PFGE-type P3XbaI; lane 7, PFGE-type P4XbaI; lane 8, PFGE-type P5XbaI; and lane 9, PFGE-type P6XbaI (8)

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Table 3 Restriction incubation calculation for enzyme XbaI Reagent

1 plug slice (μL)

10 plug slices (μL)

Sterile water

175

1,750

20

200

5

50

200

2,000

Buffer H Enzyme XbaI (10 U/μL) Total volume

Table 4 Restriction incubation calculation for enzyme AscI Reagent

1 plug slice (μL)

10 plug slices (μL)

Sterile water

174

1,740

20

200

Bovine serum albumin (BSA)

2

20

Enzyme AscI (10 U/μL)

4

40

200

2,000

Buffer 4

Total volume

Table 5 Preparation of 0.5× TBE buffer Reagent 10× TBE

Volume (mL) 100

105

110

Reagent grade water

1,900

1,995

2,090

Total volume of 0.5× TBE

2,000

2,100

2,200

9. Add 200 μL restriction enzyme mixture to each tube. Close the tube and mix by tapping gently. The plug slices should be under the enzyme mixture. 10. Incubate the plug slices in a 37 °C water bath for 4 h or longer (3–16 h) if necessary. 3.7 Casting the Agarose Gel ( See Note 14)

1. Preheat the water bath to 55–60 °C. 2. Prepare 0.5× Tris-borate EDTA buffer (TBE) for both the gel and electrophoresis running buffer according to Table 5. 3. Make 1 % SKG agarose in 0.5× TBE as follows by weighing 1 g of SKG agarose into a 500-mL screw-cap flask for a 14-cmwide gel form. Add 100 mL of 0.5× TBE and swirl gently to

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disperse agarose. For a 21-cm-wide gel form, weigh 1.5 g of SKG agarose into a 500-mL screw-cap flask. Add 150 mL of 0.5× TBE; swirl gently to disperse agarose. Loosely cover with a cap and microwave for 60 s; mix gently and repeat for 15-s intervals until agarose is completely dissolved. 4. Recap the flask and place it in a 55–60 °C water bath (see Note 15). 5. A small volume (2–5 mL) of melted and cooled (50–60 °C) 1 % SKG agarose is needed to seal the wells after the plugs are loaded. Prepare 10 mL by melting 0.1 g agarose with 10 mL of 0.5× TBE in a 100-mL screw-cap flask as described above. Unused SKG agarose can be kept at 18–25 °C, melted, and reused several times. Microwave for 15–20 s and mix; repeat for 10-s intervals until the agarose is completely melted. Place in the 55–60 °C water bath until ready to use. 6. Arrange the gel form level on a leveling table, with the front of the comb holder and teeth facing the bottom of the gel. Make sure that the comb teeth are 2 mm above the surface of the gel platform. 7. Carefully pour the agarose (cooled to 55–60 °C) into the gel form. Be sure there are no air bubbles. 8. Remove the comb after the gel solidifies for 20 min. 9. Remove the restricted plug slices from the 37 °C water bath. Remove the enzyme/buffer mixture and add 200 μL of 0.5× TBE. Incubate at 18–25 °C for 5 min. 10. Remove the plug slices from the tubes. Load the plug slices on the bottom of the comb teeth. 11. Using a spatula, load a λ ladder PFG marker and a MidRange PFG marker in the first and last wells. 12. Load the samples into the remaining wells; remove the restricted plug slices from the tubes with the tapered end of a spatula and load into the wells. Gently push the plugs to the bottom and front of the wells with the wide end of a spatula. Make sure that are no air bubbles. 13. Fill in the wells of the gel with melted 1 % SKG agarose kept at 55–60 °C. Allow to harden for 3–5 min. Remove excess buffer with a tissue. 14. Confirm that the plug slices are correctly aligned on the bottom of the comb teeth, that the lower edge of the plug slice is flushed against the black platform, and that there are no air bubbles. 15. Put the black gel frame in the electrophoresis chamber. Add 2–2.2 L of freshly prepared 0.5× TBE. Close the cover of the unit.

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16. Turn on the power supply to CHEF Mapper, pump (setting of ≈70 for a flow of 1 l/min) and cooling module (14 °C). 17. Unscrew and remove the end gates from the gel form; remove the excess agarose from the sides and bottom of the casting platform with a tissue. Keep the gel on the casting platform and carefully place the gel inside the black gel frame in the electrophoresis chamber. Close the cover of the chamber. 3.8 Electrophoresis Conditions

1. Select the following conditions on CHEF Mapper for Y. pestis plugs restricted with AscI. Auto Algorithm 25 kb—low MW 215 kb—high MW Run time = 18 h Initial switch time = 1.79 s Final switch time = 18.66 s Linear ramping factor (see Note 16) 2. Select the following conditions on the CHEF Mapper for Y. pestis plugs restricted with XbaI. Auto Algorithm 25 kb—low MW 290 kb—high MW Run time = 18 h Initial switch time = 1.79 s Final switch time = 18.66 s Linear ramping factor

3.9 Staining of the Gel

1. When the electrophoresis run is over, turn off the equipment (cooling module first then pump). Remove and stain the gel with ethidium bromide. Dilute 50 μL of ethidium bromide stock solution (10 mg/mL) with 500 mL of reagent grade water (this volume is for a staining box that is approximately 14 cm × 24 cm). Stain the gel for 20–30 min in a covered container with gentle shaking (see Note 17). 2. Wash the gel in 500 mL reagent grade water for 30 min, gently shaking on shaker. Change the water and repeat this step twice. 3. Take a picture under UV light. Save gel image as an *.img or *.1sc file; convert this file to *.tif file for analysis with BioNumerics or equivalent software. 4. Discard the buffer from the electrophoresis chamber. Rinse twice the chamber with 2 L reagent grade water.

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3.10 PFGE Data Analysis

4

1. PFGE patterns are compared using the Dice coefficient. 2. Clustering of strains is based on the unweighted pair-group method using arithmetic averages (UPGMA) with 1 % position tolerance and 1 % optimization; bands