Small things matter - Of phages and antibiotic resistance conferring plasmids

95 Pages·2016·2.63 MB·English

by  Mattila, Sari

JYVÄSKYLÄ STUDIES IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE
322
Sari Mattila
Small Things Matter
Of Phages and Antibiotic
Resistance Conferring Plasmids
JYVÄSKYLÄ STUDIES IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE 322
Sari Mattila
Small Things Matter
Of Phages and Antibiotic
Resistance Conferring Plasmids
Esitetään Jyväskylän yliopiston matemaattis-luonnontieteellisen tiedekunnan suostumuksella
julkisesti tarkastettavaksi yliopiston Ambiotica-rakennuksen salissa YAA303,
lokakuun 28. päivänä 2016 kello 12.
Academic dissertation to be publicly discussed, by permission of
the Faculty of Mathematics and Science of the University of Jyväskylä,
in building Ambiotica, hall YAA303, on October 28, 2016 at 12 o’clock noon.
UNIVERSITY OF JYVÄSKYLÄ
JYVÄSKYLÄ 2016
Small Things Matter
Of Phages and Antibiotic
Resistance Conferring Plasmids
JYVÄSKYLÄ STUDIES IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE 322
Sari Mattila
Small Things Matter
Of Phages and Antibiotic
Resistance Conferring Plasmids
UNIVERSITY OF JYVÄSKYLÄ
JYVÄSKYLÄ 2016
Editors
Varpu Marjomäki
Department of Biological and Environmental Science, University of Jyväskylä
Pekka Olsbo, Ville Korkiakangas
Publishing Unit, University Library of Jyväskylä
Jyväskylä Studies in Biological and Environmental Science
Editorial Board
Jari Haimi, Anssi Lensu, Timo Marjomäki, Varpu Marjomäki
Department of Biological and Environmental Science, University of Jyväskylä
URN:ISBN:978-951-39-6766-6
ISBN 978-951-39-6766-6 (PDF)
ISBN 978-951-39-6765-9 (nid.)
ISSN 1456-9701
Copyright © 2016, by University of Jyväskylä
Jyväskylä University Printing House, Jyväskylä 2016
ABSTRACT
Mattila, Sari
Small things matter - Of phages and antibiotic resistance conferring plasmids
Jyväskylä: University of Jyväskylä, 2016, 67 p.
(Jyväskylä Studies in Biological and Environmental Science
ISSN 1456-9701; 322)
ISBN 978-951-39-6765-9 (nid.)
ISBN 978-951-39-6766-6 (PDF)
Yhteenveto: Pienillä asioilla on merkitystä - faageista ja resistenssiplasmideista
Diss.
Viruses and plasmids are small units of genetic material dependent on cells
either transiently or continuously. Intriguingly, stories of these small entities
intertwine in antibiotic resistance crisis. Horizontal gene transfer enables
bacteria to respond rapidly to chances in their environment. Anthropogenic
consumption of antibiotics induces the travel of resistance encoding genes
mainly as passengers of conjugative plasmids. In this thesis, I demonstrate that
clinically important resistance plasmids could evolutionarily rescue susceptible
bacteria under lethal antibiotic concentrations. If mobile resistance genes are
available in surrounding community, administration of high doses of antibiotic
might not be enough to treat some bacterial infections – calling for alternatives
to fight multi-resistant bacteria and interfere the spread and maintenance of
resistance. Phage therapy, utilization of bacterial viruses against bacteria, could
be one such avenue. In this thesis plasmid-depended phage PRD1 was studied
in itself and as a tool to be utilized against resistance plasmid carrying bacteria.
Blue native polyacrylamide gel electrophoresis revealed interaction between
two virus entry related membrane proteins and zymogram analysis overruled a
previous model of the lytic enzyme residing at the genome-packaging vertex.
Bacterial resistance to PRD1 was linked with either lost or impaired conjugation
ability, that could restore only when the initial resistance-conferring mutation
was a dynamic tandem repeat insertion. Promisingly, the reversion also
returned the susceptibility to the phage. Yet, as plasmid-dependent phages are
currently available only against some resistance plasmids, an alternative
approach, on-demand isolation of phages, was investigated against common
nosocomial pathogens. Staphylococcus, Acinetobacter and Enterococcus phages
were scarce in the environmental reservoir whereas phages against E. coli, K.
pneumoniae, P. aeruginosa and Salmonella strains could often be isolated as
needed. Altogether, different manifestations of phage-therapy may provide
answers to the current antibiotic resistance crisis.

Keywords: Antibiotic resistance; bacteriophage; conjugative plasmid; horizontal
gene transfer; phage therapy; protein interactions.

Sari Mattila, University of Jyväskylä, Department of Biological and Environmental
Science, P.O. Box 35, FI-40014 University of Jyväskylä, Finland
Author’s address Sari Mattila
Department of Biological and Environmental Science
P.O. Box 35
FI-40014 University of Jyväskylä
Finland
[email protected]

Supervisors Professor Jaana Bamford
Department of Biological and Environmental Science
P.O. Box 35
FI-40014 University of Jyväskylä
Finland

Docent Matti Jalasvuori
Department of Biological and Environmental Science
P.O. Box 35
FI-40014 University of Jyväskylä
Finland

Reviewers Professor Tapani Alatossava
Department of Food and Environmental Sciences
P.O. Box 66
FI-00014 University of Helsinki
Finland

Docent Maija Pietilä
Department of Food and Environmental Sciences
P.O. Box 56
FI-00014 University of Helsinki
Finland

Opponent Professor Dennis Bamford
Department of Biosciences and Institute of Biotechnology
P.O. Box 56
FI-00014 University of Helsinki
Finland
CONTENTS

ABSTRACT
LIST OF ORIGINAL PUBLICATIONS
ABBREVIATIONS
1 INTRODUCTION ..................................................................................................... 9
1.1 Bacteriophages ................................................................................................ 11
1.2 Plasmid-dependent phage PRD1 ................................................................. 13
1.3 Bacterial resistance to antibiotics .................................................................. 15
1.4 Acquired antibiotic resistance ....................................................................... 17
1.5 Horizontal gene transfer ................................................................................ 18
1.6 Conjugative plasmids .................................................................................... 20
1.7 Beta-lactamases and the rise of ESBLs ......................................................... 22
1.8 Phage therapy.................................................................................................. 23
2 AIMS OF THE STUDY ........................................................................................... 26
3 MATERIALS AND METHODS ............................................................................ 27
4 RESULTS AND DISCUSSION .............................................................................. 28
4.1 Updating our view of protein interactions in PRD1 (I) ............................ 28
4.1.1 Cloning of gene XV for expression and purification of
GST-P15 and production of polyclonal anti-P15 ........................................ 28
4.1.2 Zymogram analysis excludes P15 from the special vertex ............ 29
4.1.3 BN-PAGE can be used to unravel protein interactions in
PRD1 particles ................................................................................................. 30
4.2 Plasmids and plasmid-dependent phages in antibiotic resistance ......... 32
4.2.1 Efficiency of evolutionary rescue via conjugation under lethal
antibiotic selection depends on plasmid type (II) ...................................... 32
4.2.2 Plasmid dependent lytic phages could be a sustainable tool
against the spread of plasmid encoded antibiotic resistances (III) ......... 37
4.3 Phage isolation success differs between bacterial host species (IV) ........ 40
5 CONCLUSIONS ...................................................................................................... 44
Acknowledgements ............................................................................................................ 45
YHTEENVETO (RÉSUMÉ IN FINNISH) .................................................................... 47
REFERENCES .................................................................................................................. 50
LIST OF ORIGINAL PUBLICATIONS
The thesis is based on the following original papers, which will be referred to in
the text by their Roman numerals I-IV

I Mattila S., Oksanen H.M. & Bamford J.K. 2015. Probing protein
interactions in the membrane-containing virus PRD1. Journal of General
Virology 96: 453–462.

II Mattila S., Ruotsalainen P., Ojala V., Tuononen T., Hiltunen T. & Jalasvuori
M. 2016. Conjugative ESBL-plasmids differ in their potential to rescue
susceptible bacteria via horizontal gene transfer. Submitted manuscript.

III Ojala V., Mattila S., Hoikkala V., Bamford J.K., Hiltunen T. & Jalasvuori M.
2016. Scoping the effectiveness and evolutionary obstacles in using
plasmid-dependent phages to fight antibiotic resistance. Future
Microbiology. 11: 999-1009.

IV Mattila S., Ruotsalainen P. & Jalasvuori M. 2015. On-demand isolation of
bacteriophages against drug-resistant bacteria for personalized phage
therapy. Frontiers in Microbiology 6: 1271.

RESPONSIBILITIES OF SARI MATTILA IN THE ARTICLES OF THE THESIS:

I I performed the experiments and prepared the tables and figures. The data
analysis and writing of the paper was done together with the co-authors.

II I isolated the plasmids and analyzed their sequence. Pilvi Ruotsalainen
and I did the PCR analysis. All the authors participated in the conjugation
experiments. I co-supervised the Master’s thesis project involving part of
these experiments. Ville Ojala and I analyzed the data from conjugation
experiments. I wrote the manuscript with the co-authors.

III I performed the sequencing. I participated in the rest of the experiments,
data analysis and writing of the article.

IV I did the experiments together with Pilvi Ruotsalainen. I prepared all the
figures and data analysis and participated in the writing of the article.
ABBREVIATIONS
ATPase adenosine triphosphatase
ATV Acidianus two-tailed virus
BN-PAGE blue native polyacrylamide gel electrophoresis
cryo-EM cryo-electron microscopy
DDM N-dodecyl-(cid:451)-maltoside
DNA deoxyribonucleic acid
ds double stranded
ESBL extended-spectrum beta-lactamase
GST glutathione S-transferase
GTAs gene transfer agents
HGT horizontal gene transfer
Inc incompatibility
kb kilobase pairs
kDa kilodalton
MGEs mobile genetic elements
MIC minimum inhibitory concentration
MPC mutant prevention concentration
Mpf mating pair formation
MRSA methicillin-resistant Staphylococcus aureus
MSW mutation selection window
OriT origin of transfer
RNA ribonucleic acid
rRNA ribosomal RNA
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
ss single-stranded
T triangulation number
TEV tobacco etch virus
T4CP type IV coupling protein
wt wild-type