Table Of ContentMethods in
Molecular Biology 1157
Kieran Jordan
Edward M. Fox
Martin Wagner Editors
Listeria
monocytogenes
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
h ttp://www.springer.com/series/7651
Listeria monocytogenes
Methods and Protocols
Edited by
Kieran Jordan
Teagasc Food Research Centre, Fermoy, Cork, Ireland
Edward M. Fox
Food Microbiology and Safety Group, Animal, Food and Health Sciences,
CSIRO, Werribee, VIC, Australia
Martin Wagner
University of Veterinary Medicine Vienna, Vienna, Austria
Editors
Kieran J ordan Edward M. F ox
Teagasc Food Research Centre Food Microbiology and Safety Group
Fermoy, Cork , Ireland Animal, Food and Health Sciences, CSIRO
Werribee, VIC, Australia
Martin W agner
University of Veterinary Medicine Vienna
Vienna, Austria
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-0702-1 ISBN 978-1-4939-0703-8 (eBook)
DOI 10.1007/978-1-4939-0703-8
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Prefa ce
From its fi rst description by Murray et al. in 1926 (referred to as B acterium monocytogenes ),
Listeria monocytogenes has frequently been associated with infection of humans and animals
[1, 2]. The dual lifestyle of L . monocytogenes , from environmental saprophyte to pathogen,
has sparked interest in scientists across a range of fi elds and has advanced our understanding
of the biology of the bacterium [3]. The evolution of this understanding has been charac-
terized by many notable milestones. Studies on the ecology of L. monocytogenes illustrated
its ubiquitous nature, with a large range of environments harboring the organism, includ-
ing soil, plant material, water, and wastewater, to carriage by animals and humans, often
asymptomatically [4, 5]. Although current knowledge suggests that cases of human listerio-
sis are almost exclusively through foodborne infection, this critical transmission vector
became clear only during the 1980s, largely the result of a series of high-profi le disease
outbreaks, perhaps most notable of which was the Canadian outbreak of 1981, linked to
contaminated coleslaw [6]. With many foodborne outbreaks recorded globally every year
since then, some of which have been amongst the most severe of any attributed to a bacte-
rial pathogen [7, 8], L. monocytogenes has been a driving force in the development of cur-
rent disease surveillance and control strategies. This includes global surveillance networks
such as PulseNet, which allows international comparison of different strains of L. monocyto-
genes . Along with these advances in the epidemiology of the organisms, other strides were
being made in the understanding of the pathogenesis of the organism, including its intra-
cellular nature and how this contributed to crossing three key barriers—the intestinal bar-
rier, the blood–brain barrier, and also the fetoplacental barrier, perhaps most characteristic
of this pathogen [9, 10, 11]. The knowledge of this intricate mode of infection has led to
the recent reformation of the interaction between L . monocytogenes and humans, which has
seen the agent of one of the most severe bacterial diseases of humans being used in the fi ght
against cancer, one of the leading causes of human mortality [12, 13].
This long journey in the understanding of L. monocytogenes has been achieved through
a vast array of research covering a wide range of scientifi c areas, including, in recent years,
molecular methodologies. These achievements have often been made through innovative
strategies devised to address many different questions regarding the biology of the organ-
ism, from pathogenicity and virulence to characterization and tracking sources, and are
characterized by the development of many scientifi c methodologies.
Methods in Molecular Biology is a series of books that presents a step-by-step protocol
approach to experimentation. Each protocol opens with an introductory overview, a list of
the materials and reagents needed to complete the experiment, and is then followed by a
detailed procedure supported with a notes section offering tips and tricks of the trade as
well as troubleshooting advice. The protocols are comprehensive and reliable.
As Listeria monocytogenes continues to be a major threat to public health, this book in
the series is a timely addition. It brings together protocols and methodologies that are used
in research to gain a better understanding of L isteria at a molecular level. The topics covered
include sampling in order to isolate Listeria , methods for their identifi cation and
v
vi Preface
characterization, methods for gene manipulation, and fi nally methods for the control of the
organism. The book will contribute towards the harmonization of the methods used and
will therefore benefi t all those interested in Listeria research.
Fermoy, Ireland Kieran Jordan
Werribee, VIC, Australia Edward M. Fox
Vienna, Austria Martin Wagner
References
1 . M urray EGD, Webb RA, Swann MBR (1926) Hayes P, Weaver R, Audurier A, Plikaytis BD,
A disease of rabbits characterized by large Fannin SL, Kleks A, Broome CV (1988)
mononuclear leucocytosis, caused by a hitherto Epidemic listeriosis associated with Mexican-
undescribed bacillus, B acterium monocytogenes style cheese. N Engl J Med 319:823–828
(n. sp.). J Pathol Bacteriol 29:407–439 8 . B ille J (1990) Epidemiology of human liste-
2 . G ray ML, Killinger AH (1966) L isteria mono- riosis in Europe with special reference to the
cytogenes and listeric infections. Bacteriol Rev Swiss outbreak. In: Miller AJ, Smith JL,
30:309–382 Somkuti GA (eds) Foodborne listeriosis.
3 . F reitag NE, Port GC, Miner MD (2009) Society for Industrial Microbiology. Elsevier
Listeria monocytogenes —from saprophyte to Science Publishing, Inc., New York, pp
intracellular pathogen. Nat Rev Microbiol 7:623 71–74
4 . M cCarthy SA (1990) Listeria in the environ- 9. C hakraborty T, Goebel W (1988) Recent
ment. In: Miller AJ, Smith JL, Somkuti GA developments in the study of virulence in
(eds) Foodborne listeriosis. Society for Listeria monocytogenes . Curr Top Microbiol
Industrial Microbiology. Elsevier Science Immunol 138:41–48
Publishing, Inc., New York, pp 25–29 10. L ecuit M (2005) Understanding how Listeria
5 . G rif K, Patscheider G, Dierich MP, Allerberger monocytogenes targets and crosses host barri-
F (2003) Incidence of fecal carriage of Listeria ers. Clin Microbiol Infect 11:430–436
monocytogenes in three healthy volunteers: a 1 1. S eveau S, Pizarro-Cerda J, Cossart P (2007)
one-year prospective stool survey. Eur J Clin Molecular mechanisms exploited by Listeria
Microbiol Infect Dis 22:16–20 monocytogenes during host cell invasion.
6 . S chlech WF III, Lavigne PM, Bortolussi RA, Microbes Infect 9:1167–1175
Alien AC, Haldane EV, Wort AJ, Hightower 12. R othman J, Paterson Y (2013) Live-attenuated
AW, Johnson SE, King SH, Nicholls ES, Listeria -based immunotherapy. Expert Rev
Broome CV (1983) Epidemic listeriosis-evi- Vaccines 12:493–504
dence for transmission by food. N Engl J Med 13. Le DT, Dubenksy TW Jr, Brockstedt DG
308:203–206 (2012) Clinical development of Listeria mono-
7 . L innan MJ, Mascola L, Lou XD, Goulet V, cytogenes -based immunotherapies. Semin
May S, Salminen C, Hird DW, Yonekura ML, Oncol 39:311–322
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I DETECTION, QUANTIFICATION, AND CONFIRMATION
1 Sampling the Processing Environment for Listeria. . . . . . . . . . . . . . . . . . . . . . 3
Anca Ioana N icolau and Andrei Sorin B olocan
2 T raditional Methods for Isolation of Listeria monocytogenes. . . . . . . . . . . . . . . 15
Rui M agalhães, C ristina M ena, V ânia Ferreira,
Gonçalo Almeida, Joana S ilva, and P aula Teixeira
3 C onfirmation of Isolates of Listeria by Conventional
and Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1
David R odríguez-Lázaro and M arta H ernández
PART II CHARACTERIZATION AND TYPING
4 Serotype Assignment by Sero-Agglutination, ELISA, and PCR . . . . . . . . . . . . 4 1
Lisa G orski
5 P ulsed-Field Gel Electrophoresis (PFGE) Analysis
of Listeria monocytogenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Marion D almasso and K ieran J ordan
6 M ultilocus Sequence Typing (MLST) of Listeria monocytogenes. . . . . . . . . . . . . 73
Beatrix S tessl, I rene Rückerl, and Martin Wagner
7 Ribotyping and Automated Ribotyping of Listeria monocytogenes. . . . . . . . . . . 85
Mazin Matloob and Mansel G riffiths
8 F luorescent Amplified Fragment Length Polymorphism (fAFLP)
Analysis of Listeria monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5
Corinne A mar
9 High-Throughput Characterization of Listeria monocytogenes
Using the OmniLog Phenotypic Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Edward M . Fox and K ieran J ordan
1 0 Analysis of Listeria monocytogenes Subproteomes. . . . . . . . . . . . . . . . . . . . . . . 109
Michel Hébraud
11 The Listeria Cell Wall and Associated Carbohydrate Polymers. . . . . . . . . . . . . 1 29
Marcel R. E ugster and Martin J . L oessner
12 U se of Bacteriophage Cell Wall-Binding Proteins
for Rapid Diagnostics of Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 41
Mathias S chmelcher and Martin J. L oessner
vii
viii Contents
13 Virulence Characterization of Listeria monocytogenes. . . . . . . . . . . . . . . . . . . . . . 1 57
Swetha R eddy and M ark L. L awrence
14 I nternalization Assays for Listeria monocytogenes . . . . . . . . . . . . . . . . . . . . . . . 1 67
Andreas K ühbacher, P ascale C ossart, and J avier P izarro-Cerdá
PART III STRAIN MANIPULATION
15 Extraction and Analysis of Plasmid DNA from Listeria monocytogenes . . . . . . . 1 81
Aidan Casey and O livia M cAuliffe
16 Generation of Nonpolar Deletion Mutants in Listeria monocytogenes
Using the “SOEing” Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 87
Kathrin R ychli, C aitriona M . G uinane, Karen D aly,
Colin Hill, and Paul D . C otter
17 M utant Construction and Integration Vector-Mediated Gene
Complementation in Listeria monocytogenes. . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Reha Onur A zizoglu, D riss E lhanafi, and Sophia K athariou
18 Absolute and Relative Gene Expression in Listeria monocytogenes
Using Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 13
Roberta Mazza and Rina M azzette
19 Genome Sequencing of Listeria monocytogenes. . . . . . . . . . . . . . . . . . . . . . . . . 223
Stephan Schmitz-Esser and Martin W agner
20 U sing Enhanced Green Fluorescent Protein (EGFP) Promoter Fusions
to Study Gene Regulation at Single Cell and Population Levels. . . . . . . . . . . . 233
Marta Utratna and Conor P. O ’Byrne
PART IV CONTROL METHODS
21 Control of Listeria monocytogenes in the Processing Environment
by Understanding Biofilm Formation and Resistance to Sanitizers. . . . . . . . . . 251
Stavros G. Manios and Panagiotis N. Skandamis
22 V accination Studies: Detection of a Listeria monocytogenes- Specific
T Cell Immune Response Using the ELISPOT Technique . . . . . . . . . . . . . . . 263
Mohammed Bahey-El-Din and Cormac G .M. G ahan
23 S ampling the Food Processing Environment: Taking Up the Cudgel
for Preventive Quality Management in Food Processing Environments . . . . . . 275
Martin Wagner and Beatrix S tessl
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 85
Contributors
GONÇALO A LMEIDA • Laboratório Associado, Escola Superior de Biotecnologia,
Universidade Católica Portuguesa/Porto , Porto , P ortugal
CORINNE AMAR • Gastrointestinal Bacteria Reference Unit , P ublic Health England ,
London, U K
REHA ONUR AZIZOGLU • Department of Food, Bioprocessing and Nutrition Sciences ,
North Carolina State University , Raleigh, NC, U SA
MOHAMMED BAHEY-EL-DIN • Department of Pharmaceutical Microbiology,
Faculty of Pharmacy, A lexandria University , A lexandria, Egypt
ANDREI SORIN BOLOCAN • Faculty of Food Science and Engineering, D unarea de Jos
University of Galati , Galati, Romania
AIDAN CASEY • Teagasc Food Research Centre , Fermoy, Cork, I reland
PAUL D . COTTER • Teagasc Food Research Centre , F ermoy, Cork, Ireland
PASCALE COSSART • Institut Pasteur, Unité des Interactions Bactéries Cellules , Paris, F rance
MARION DALMASSO • Teagasc Food Research Centre , F ermoy, Cork, I reland
KAREN D ALY • Department of Microbiology, U niversity College Cork , C ork , I reland
DRISS ELHANAFI • Biomanufacturing Training and Education Center , N orth Carolina
State University , Raleigh, NC, U SA
MARCEL R. EUGSTER • Institute of Food, Nutrition and Health, ETH Zurich ,
Zurich, S witzerland
VÂNIA F ERREIRA • Laboratório Associado, Escola Superior de Biotecnologia,
Universidade Católica Portuguesa/Porto , P orto , P ortugal
EDWARD M. FOX • Food Microbiology and Safety Group , Animal, Food and Health Sciences,
CSIRO , W erribee, VIC , A ustralia
CORMAC G . M. GAHAN • Department of Microbiology and School of Pharmacy ,
University College Cork , Cork, I reland ; A limentary Pharmabiotic Centre, University
College Cork, C ork , I reland
LISA G ORSKI • Produce Safety and Microbiology Research Unit, United States Department
of Agriculture , A gricultural Research Service , Albany, C A , U SA
MANSEL G RIFFITHS • Canadian Research Institute for Food Safety, University of Guelph ,
Guelph , O N , C anada
CAITRIONA M. GUINANE • Teagasc Food Research Centre , F ermoy, C ork , I reland
MICHEL H ÉBRAUD • INRA, Clermont-Ferrand Research Centre , U R454 Microbiology ,
Saint-Genès Champanelle, France
MARTA HERNÁNDEZ • Instituto Tecnológico Agrario (ITACyL) , V alladolid, Spain
COLIN HILL • Department of Microbiology, U niversity College Cork , Cork, I reland
KIERAN JORDAN • Teagasc Food Research Centre , Fermoy, C ork , I reland
SOPHIA KATHARIOU • Department of Food, Bioprocessing and Nutrition Sciences and
Biomanufacturing Training and Education Center , North Carolina State University ,
Raleigh , N C, USA
ANDREAS KÜHBACHER • Institut Pasteur, Unité des Interactions Bactéries Cellules ,
Paris , F rance
ix
