Stress responses of Listeria monocytogenes to antimicrobial

176 Pages·2010·4.57 MB·English

Stress responses of Listeria monocytogenes to antimicrobial
substances from lactic acid bacteria: an explorative study

Stressresponser hos Listeria monocytogenes ovenfor antimikrobielle substanser fra
melkesyrebakterier: et eksplorativt studium

Philosophiae Doctor (PhD) Thesis

Girum Tadesse Tessema

Ås 2010

Norwegian University of Life Sciences
Department of Chemistry, Biotechnology and Food Science

Thesis number 2011:02
ISBN 978-82-575-0960-6
ISSN 1503-1667

Faith is to believe what you do not see;
the reward of this faith is to see what you believe.
St. Augustine (354-430)

TABLE OF CONTENTS

ACKNOWLEDGMENTS.......................................................................................................ii
ABSTRACT.............................................................................................................................iii
SAMMENDRAG (NORWEGIAN ABSTRACT)................................................................iv
LIST OF PAPERS....................................................................................................................v
1. INTRODUCTION................................................................................................................1
2. BACKGROUND...................................................................................................................3
2.1 Listeria monocytogenes....................................................................................................3
2.2 Significance of L. monocytogenes and human listeriosis.................................................3
2.2.1 Pathogenicity and virulence determinants.................................................................4
2.3 Food safety and biopreservatives.....................................................................................5
2.3.1 Lactic acid bacteria and their antimicrobial products...............................................6
2.4 Stress responses of L. monocytogenes............................................................................12
2.4.1 Response to the class IIa bacteriocin stress.............................................................12
2.4.2 Response to acid stress............................................................................................16
2.4.3 Effect of class IIa bacteriocins and low pH stresses on virulence...........................21
2.5 Methodological aspects of the present stress response study.........................................22
2.5.1 Fourier transform infrared spectroscopy.................................................................23
2.5.2 Transcriptional analysis...........................................................................................24
2.5.3 Data analysis...........................................................................................................26
3. OBJECTIVES OF THE STUDY......................................................................................27
4. MAIN RESULTS AND DISCUSSION............................................................................28
5. MAIN CONTRIBUTION AND FUTURE WORK.........................................................37
6. REFERENCES...................................................................................................................38
7. APPENDIX.........................................................................................................................54
Paper I-III

ACKNOWLEDGMENTS

The work presented in this thesis was carried out at Nofima Mat AS and financially supported
by The Foundation for Research Levy on Agricultural Products.

I am grateful to my supervisors Dr. Trond Møretrø, Dr. Kristine Naterstad, and Dr. Lars
Axelsson at Nofima Mat. Thank you for accepting me as your student and for your
unconditional encouragements and help. I also thank Professor Ingolf Nes, my supervisor at
the Norwegian University of Life Sciences, for the support with administrative works and the
heartening spirits. I would like to thank all the co-authors for the educative and productive
collaborations.

I thank all colleagues at Nofima Mat for the support and the pleasant time. Especially
enormous gratitude to past and present members of the “microbiology group” and officemates
for the unreserved guidance. I wish to extend my extraordinary gratefulness to “my parents”
Professor Arnfinn Sundsfjord and Dr. Kristin Hegstad Dahl for bringing me to the world of
molecular microbiology. Dear Arnfinn and Kristin I will cherish you fondly forever.

I also love to extend my deepest appreciation to all my families and friends for the
unconditional supports. Thanks to Emahoye, my sisters, brothers, nephews, and nieces
(yekerta besem endaltera bezu nachu☺) and your keen interest have been a great inspiration.
Mojej rodzinie w Polsce dziękuję bardzo! Mom and Dad, thanks for your endless efforts to
inculcate in me the values of education, hard work and honesty. I am just beginning to
ascertain your dreams.

My dearest wife Basia and our charismatic daughter Marta, thank you for the wonderful love
and you are the best gift ever. I am so grateful for your patience and uplifting care. Without
you the PhD journey would remain just a dream. Finally, I wish to thank all those who have
contributed in one way or another, especially, God for His indescribable gift!!!

Ås, October 2010

Girum Tadesse Tessema
ii

ABSTRACT

Listeria monocytogenes is a gram-positive bacterium that causes severe and life threatening
disease both in humans and animals. Due to the severity of the disease and the fact that the
bacterium is responsible for considerable economical loss, L. monocytogenes is of a great
concern particularly to the food industry. L. monocytogenes is a common food contaminant
and nearly all human L. monocytogenes infections are due to ingestion of contaminated food.
Antimicrobial components from lactic acid bacteria may serve as safe and natural food
preservatives targeting unwanted microorganisms, including L. monocytogenes. Elucidations
of how L. monocytogenes respond to the antimicrobial products are crucial steps to devise a
knowledge-based strategy to control this deadly bacterium in food and food-related
environments using food grade bacteria.

In the present study the responses of L. monocytogenes to the class IIa bacteriocin sakacin P
and to acids (acetic, lactic and hydrochloric acid) were explored. Exposure to sakacin P gave
spontaneous mutant strains with reduced susceptibility to the bacteriocin. Analysis of large
number of the mutant strains using different approaches revealed substantial difference among
the strains. The mutant strains displayed difference in (i) the level of resistance to sakacin P,
(ii) the stability of the sakacin P resistance phenotype, (iii) growth fitness in various
conditions, (iv) biofilm formation ability, (v) virulence potential, (vi) Fourier transform
infrared spectroscopy profile, (vii) regulation of the bacteriocin receptor gene, and (viii)
global transcriptome profile. Overall, this indicates that the incidence(s) giving rise to the
sakacin P resistance involves a complex regulatory gene network possibly mediated by the
bacteriocin receptor and have pleiotropic effects on the physiology of the resistant strains.

The growth of L. monocytogenes was reduced but not completely inhibited at pH 5 when the
growth medium was acidified with hydrochloric acid (HCl), 10 mM acetic acid or 20 mM
lactic acid. Acetic acid had the highest antilisterial activity followed by lactic acid and HCl.
Stress due to the presence of the acids induced a large number of genes associated with acid
defense, virulence, and cross-protection to other types of stresses. Acidulant type dependent
responses were also observed.

The explorative transcriptome studies confirmed numerous results of previous studies on the
response of L. monocytogenes to the class IIa bacteriocins and acid stresses. In addition, it
identified a number of putative genes with possible role in the responses under investigation.
Altogether, the results presented in this thesis revealed insights contributing to understand the
responses of L. monocytogenes to the antimicrobial substances that may be encountered by
this bacterium in fermented food and thereby opening new avenues for further studies.

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