Antimicrobial screening of secondary metabolites from Solanaceae Katarina Jane Nice

319 Pages·2013·12.52 MB·English

by  Sebastian Katie

Antimicrobial screening of secondary
metabolites from Solanaceae
Katarina Jane Nice
This thesis was submitted for the degree of Doctor of Philosophy at Royal
Holloway University of London, in September 2012
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Candidate declaration
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Candidate declaration
I declare that the work presented in this thesis is the original work of the author unless
otherwise stated. Original material used in the creation of this thesis has not been
previously submitted either in part or whole for a degree of any description from any
institution.
Katarina Nice
……………………………………..
Katarina Nice
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Abstract
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Abstract
The aim of the project was to analyse the potential antimicrobial activity of extracted
secondary metabolites from wild tomato species, and other Solanaceous plants. The
wild tomato species such as Solanum lycopersicum cv M82, Solanum cheesmaniae,
Solanum chmielewskii, Solanum chilense, Solanum peruvianum, Solanum
pimpinellifolium, Solanum pennellii, Solanum habrochaites, and Solanum neorickii
were the primary focus of the project which were analysed for antimicrobial
compounds. The hypothesis that the wild tomato relatives could be antimicrobial is
linked to the insecticidal and fungicidal properties of several wild tomato relatives. This
research is further supported by the previous findings into the antimicrobial potential of
certain Solanaceae species, where the leaf tissue was noted to have the highest general
antimicrobial activity.
As part of this project the fully expanded undamaged leaves were examined for
antimicrobial activity, after extracting the secondary metabolites in a range of solvents.
Antimicrobial activity was determined through monitoring the plant extracts effect on
the growth of gastroenteric bacteria Salmonella, Escherichia coli, and Staphylococcus
aureus primarily through disc diffusion assays and growth curve analysis. The
metabolomic profile of Staphylococcus aureus grown in the presence of identified
antimicrobial Solanum pennellii extract was used to identify the compounds mode of
action, and electron microscopy was used to view the effects of this extract upon the
bacteria cell structure.
Further putative identification of selected antimicrobial extracts was achieved through
the use of thin layer chromatography, and gas chromatrography-mass spectrometry
analysis. The Solanaceae plants which were identified to have a significant
antimicrobial activity were Solanum habrochaites, Solanum pennellii, and Nicotiana
rustica. The trichomes of these plants were identified as the primary target site for the
production of these antimicrobial compounds.
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Acknowledgements
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Acknowledgements
I would like to thank all my family and friends for their continued support and
motivation during this period of my life. Particularly my best friend Emily Shell who is
always there for me when I need her, my mother Jane Head for her enthusiastic
encouragement, and my husband Sebastian Nice for his love and support.
I would like to thank all my colleagues at the AHVLA and the RHUL who have aided
me in this research, and who have trained me in specific laboratory techniques. With
special thanks to Mary Bagnall, Emma Kennedy, Chris Gerrish, Dr. Daniel Rickett, Dr.
Laura Perez-Fons, Dr. Michael Goodfellow, Dr. Matthew Jones, Dr. Leticia Mora, and
Dr. Tom Wells, for your help and support.
I would also like to thank Bill Cooley for performing the EM work, and I would like to
thank Prof. Graham Seymour at the University of Nottingham for initially supplying
plant material.
I could not have completed this project without the financial support and resources from
the Animal Health and Veterinary Laboratories Agency and the Royal Holloway which
included a Thomas Holloway Scholarship.
My supervisors have been fantastic, and I would like to thank them all sincerely; Prof.
Roberto La Ragione for his sensible guidance, Prof. Peter Bramley for his advice and
support, and particularly Dr. Paul Fraser for his confidence in me, and for allowing me
to complete this research with creative freedom.
I thank you all.
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Dedication
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Dedication
This thesis is dedicated to my husband Sebastian Nice, because he has dedicated his life
to me.
In memory of Anakin.
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List of Abbreviations
Abs Absorbance
ARB Antibiotic resistant bacteria
ANOVA Analysis of variance
AUC Area under the curve
BCFAs Branched-chain fatty acids
C Carbon
c Celsius
Ca Chlorophyll a
Cb Chlorophyll b
CDT Cytolethal distending toxin
CBTols Cembratrienols
CFU Colony forming unit
CI Confidence interval
CMV Cucumber mosaic virus
cv Cultivar
DPPC Dipalmitoylphosphatidycholine
DHEC Diarrhoea-associated haemolytic
DMS Dimethyl sulfide
DMSO Dimethyl sulfoxide
DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase
DNA Deoxyribonucleic acid
DW Dry weight
EPEC Enteropathogenic E. coli
ESBIs Extended spectrum beta lactamases
EHEC Enterohaemorrhagic E. coli
EIEC Enteroinvasive E. coli
ETEC Enterotoxigenic E. coli
EaggEC Enteroaggregative
ECM Extracellular matrix
EM Electron microscopy
EPS Exopolysaccharides
ELISA Enzyme-linked immunosorbent assay
FW Fresh weight
GC-MS Gas chromatography-mass spectrometry
HCL Hydrochloric acid
HPLC-PDA High performance liquid chromatography-photodiode array
HPLC High performance liquid chromatography
h Hour
HUS Haemolytic uremic syndrome
KPT Potassium phosphotungstate
ILs Introgression lines
L Litre
LB Luria broth
LPS Lipopolysaccharide
LC-MS Liquid chromatography-mass spectrometry
LC-PDA-TOFMS Liquid chromatography-photodiode-array-time of flight-mass
spectrometry
MDR Multi drug resistant
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