Investigating antimicrobial resistance in the gut bacteria of insects feeding on plants

234 Pages·2017·3.86 MB·English

by  Katarzyna Ignasiak

Investigating antimicrobial
resistance in the gut
bacteria of insects feeding
on plants
Katarzyna Bronisława Ignasiak
This thesis has been submitted in partial fulfilment of the requirements of the degree
of Doctor of Philosophy at the University of East Anglia
Department of Biological Chemistry
John Innes Centre
Norwich
June 2016
© This copy of the thesis has been supplied on condition that anyone who consults it is
understood to recognise that its copyright rests with the author and that no quotation
from the thesis, nor any information derived therefrom, may be published without the
author's prior, written consent.
Statement
The work submitted within this thesis is entirely my own, except where due reference
has been paid, and has not be submitted to this or any other university as part of any
degree.
I’m very highly educated. I know words, I have the best words.
D. J. T.
Abstract
It has been previously described that antibiotic-resistant bacteria can be found in the
guts of insects feeding on a variety of plants and not exposed to significant levels of
antibiotics. Such naturally-occurring resistance has implications for clinically-relevant
antibiotic resistance, which is a worldwide problem, and for using plants as a source of
potential novel antibiotics. We investigated this phenomenon further. Firstly, we
searched for antibiotic resistance in different insects’ guts and explored its origin, using
two lepidopteran hosts feeding on artificial food containing either ciprofloxacin or
oxytetracycline. We discovered that these antibiotics have a diverse impact on the
insect gut microbiome, beyond simply inducing antibiotic resistance. Secondly, we used
the insect gut bacteria to identify plant extracts with antibacterial activity, and purified
their active fractions. We found that vindoline, from leaf extract, and serpentine, from
root extract, are the most abundant metabolites in active fractions of Madagascar
periwinkle extracts. Finally, we developed one of the insect species we used, Galleria
mellonella, into a laboratory model for antibiotic efficacy testing, toxicity testing and as
a model for human baby gut. In summary, in this project we explored different aspects
of the antibiotic resistance that can be found in the insect gut and used it to guide us
towards plant metabolites with antibacterial properties.
Acknowledgements
I would like to thank my supervisor Prof. Tony Maxwell for his support and
encouragement. If I could start my project all over again, I would change a lot of things,
but definitely not my supervisor. I would also like to thank my supervisory committee:
Dr Janneke Balk, Dr Nicolas Burton, Dr Martin Rejzek and Dr Martin Stocks, for their
advice and constructive criticism.
I would like to thank the members of the JIC Entomology Facility: Dr Ian Bedford, Anna
Jordan, Gavin Hatt and Jake Stone, and Metabolite Services: Dr Lionel Hill, Paul Brett
and Baldeep Kular. Their assistance was irreplaceable and advice priceless.
I would like to thank Lesley Mitchenall and other members of the Maxwell lab for such
a positive working environment. Even though I was the odd one out not working on
topoisomerases, they made me feel like a true member of the group. Additionally, I
would like to thank the undergraduate students I have supervised: Aristide Lebreton,
Ryan Hull and George Newell, who I hope learned as much from me as I have learned
from them.
On a more personal note, I would like to thank my parents, Ewa and Kazimierz Ignasiak,
and my wonderful twin Marta Ignasiak. They keep motivating me and support me
through anything, even creating radioactive cauliflowers, which they thought I worked
on.
I wouldn’t have worked as hard without the encouragement from my husband Sigurjón
Ingólfsson. His long working hours and lack of work/life balance made working towards
a PhD feel like a holiday. I am grateful to have him by my (left) side, even when he is
5,000 miles away.
Finally, I would like to thank all my friends, especially the ones I have met in Norwich:
Jenny, Emma, Farzana, Inga, Tash, James, Andrew and Jorge. They all smell and it was a
great pleasure to be mean to them.
Table of Contents
1 Introduction....................................................................................................................1
1.1 Plant secondary metabolites..................................................................................1
1.1.1 Classes of plant secondary metabolites..........................................................1
1.1.2 The use of plants in traditional medicine........................................................5
1.1.3 The biological roles of plant secondary metabolites......................................7
1.2 Insects.....................................................................................................................7
1.2.1 Insect anatomy................................................................................................7
1.2.2 The role of insects...........................................................................................8
1.2.3 Insect diversity..............................................................................................10
1.2.4 Insect immunity.............................................................................................10
1.2.5 Similarities between insect and human immunity.......................................11
1.3 Insects in research context...................................................................................12
1.3.1 Insect species used in immunity studies.......................................................12
1.3.2 Using insects as scientific tools.....................................................................13
1.3.3 Interactions between plants and insects......................................................14
1.3.4 Bacteria can mediate interactions between insects and plants...................14
1.4 Microbiome diversity............................................................................................15
1.4.1 Studying bacteria: from culturing single isolates to population studies.......15
1.4.2 Microbiome composition and function........................................................16
1.4.3 Functional verification of the roles of microbiome.......................................17
1.5 Microbiology.........................................................................................................19
1.5.1 Antibiotics and antibiotic resistance.............................................................19
1.5.1.1 The history of antibiotics.......................................................................19
1.5.1.2 The emergence of antibiotic resistance................................................21
1.5.1.3 Factors contributing to antibiotic resistance.........................................22
1.5.2 Antibiotic resistance in the ecological context..............................................23
1.5.2.1 The origin of antibiotic resistance.........................................................23
1.5.2.2 The role of antibiotic resistance in ecosystems.....................................24
1.5.3 Antibiotic resistance in the context of human antibiotic use.......................25
1.5.3.1 Significance of antibiotic resistance......................................................25
1.5.3.2 Antibiotic discovery gap........................................................................25
1.5.3.3 Reasons behind the discovery void.......................................................26
1.5.3.4 Potential solutions.................................................................................28
1.5.3.5 Rational approach to plant screening for antibiotic discovery.............30
1.5.3.6 Successful plant-derived medicines......................................................31
1.6 What can we learn from the insect gut bacteria?................................................32
1.7 Summary and aims................................................................................................33
2 Materials and methods.................................................................................................35
2.1 Media and solutions..............................................................................................35
2.1.1 Solutions........................................................................................................35
2.1.2 Media............................................................................................................35
2.1.3 Test compounds............................................................................................36
2.2 Insect methods......................................................................................................39
2.2.1 Insect husbandry...........................................................................................39
2.2.2 Artificial food.................................................................................................39
2.2.3 Collection of insects......................................................................................40
2.2.4 Insect dissection............................................................................................40
2.2.5 Experimental procedures..............................................................................42
2.3 Microbiology.........................................................................................................48
2.3.1 General procedure for identifying insect gut bacteria..................................48
2.3.2 Methods used for Galleria mellonella...........................................................52
2.3.3 Metagenomic analyses..................................................................................54
2.3.4 Antibiotic susceptibility testing.....................................................................56
2.4 Plant extracts........................................................................................................56
2.4.1 Plant extract preparation..............................................................................56
2.4.2 Antibacterial activity testing.........................................................................57
2.4.3 Activity-guided fractionation........................................................................57
2.4.4 Mass spectrometry........................................................................................58
3 The impact of antibiotics on the gut microbiota of two lepidopteran hosts: Plutella
xylostella and Galleria mellonella....................................................................................61
3.1 Abstract.................................................................................................................61
3.2 Introduction..........................................................................................................62
3.2.1 Antibiotic resistance is ancient.....................................................................62
3.2.2 Antibiotic resistance is widespread...............................................................62
3.2.3 Antibiotic resistance is present in the insect gut..........................................63
3.2.4 Insects as vectors mediating the spread of antibiotic resistance.................64
3.2.5 Implications of the presence of antibiotics in insect diets............................65
3.2.6 Objectives......................................................................................................65
3.3 Results...................................................................................................................66
3.3.1 Plutella xylostella feeding on artificial food with ciprofloxacin....................66
3.3.1.1 Diamondback moth (Plutella xylostella)................................................66
3.3.1.2 Gut bacteria isolated from P. xylostella exposed to ciprofloxacin.........66
3.3.1.3 Antibiotic resistance in the gut bacteria of P. xylostella larvae ............69
3.3.2 Plutella xylostella feeding on artificial food with oxytetracycline.................71
3.3.2.1 Re-design of the experimental procedure.............................................71
3.3.2.2 Identification of bacteria from the guts of P. xylostella larvae feeding on
oxytetracycline...................................................................................................72
3.3.2.3 Antibiotic resistance in the gut bacteria of P. xylostella larvae.............72
3.3.3 Galleria mellonella feeding on artificial food with oxytetracycline..............73
3.3.3.1 Greater wax moth (Galleria mellonella)................................................73
3.3.3.2 Continuation of the experiment designed for P. xylostella...................74
3.3.3.3 Feeding procedure.................................................................................75
3.3.3.4 Isolation of gut bacteria........................................................................77
3.3.3.5 Identification of tet resistance elements from the G. mellonella guts.77
3.3.3.6 Metagenomic analysis of the G. mellonella gut contents.....................80
3.3.3.7 Species identified from the guts of G. mellonella.................................81
3.4 Discussion..............................................................................................................84
3.4.1 P. xylostella feeding on artificial food with ciprofloxacin or oxytetracycline84
3.4.1.1 Comparison of bacteria cultured...........................................................84
3.4.1.2 The lack of ciprofloxacin and oxytetracycline resistance in the guts of P.
xylostella larvae feeding on these antibiotics...................................................85
3.4.1.3 Resistance to other antibiotics in the guts of P. xylostella feeding on
ciprofloxacin and oxytetracycline......................................................................85
3.4.2 G. mellonella feeding on artificial food with oxytetracycline.......................86
3.4.2.1 Bacteria cultured from the guts of G. mellonella..................................86
3.4.2.2 Metagenomic analysis...........................................................................88
3.4.2.3 Survey of tet resistance genes in G. mellonella gut..............................88
3.4.3 Limitations of the study................................................................................89
3.4.3.1 The use of type strains as experimental controls..................................89
3.4.3.2 Low numbers of bacterial strains recovered from the insect guts........89
3.4.3.3 Comments about artificial food.............................................................90
3.4.4 Summary and conclusions.............................................................................91
4 Investigating antibiotic resistance in the insect gut bacteria: the search for novel
plant-derived antibacterials.............................................................................................93
4.1 Abstract.................................................................................................................93
4.2 Introduction..........................................................................................................94
4.2.1 Antibiotic discovery void...............................................................................94
4.2.2 Phytochemicals as novel antimicrobials........................................................95
4.2.3 Food with antibacterial activity can lead to antibiotic resistance in the insect
gut..........................................................................................................................97
4.2.4 Aims of this chapter......................................................................................98
4.3 Results...................................................................................................................99
4.3.1 Giant stick insect (Diapherodes gigantea) feeding on eucalyptus (Eucalyptus
dalrympleana)........................................................................................................99
4.3.1.1 Diapherodes gigantea............................................................................99
4.3.1.2 Gut dissections....................................................................................100
4.3.1.3 Identification of the gut bacteria........................................................101
4.3.1.4 Antibiotic susceptibility testing...........................................................102
4.3.1.5 16S metagenomic analysis..................................................................103
4.3.1.6 Eucalyptus extract preparation...........................................................108
4.3.1.7 Eucalyptus extract fractionation.........................................................110
4.3.2 Diamondback moth (Plutella xylostella) feeding on cabbage (Brassica rapa)
..............................................................................................................................114
4.3.2.1 Plutella xylostella.................................................................................114
4.3.2.2 Gut dissections....................................................................................116
4.3.3 Cinnabar moth (Tyria jacobaeae) feeding on ragwort (Jacobaea vulgaris).118
4.3.3.1 Cinnabar moth.....................................................................................118
4.3.3.2 Gut bacteria.........................................................................................120
4.3.3.3 Antibiotic resistance............................................................................120
4.3.3.4 Ragwort extract...................................................................................122
4.3.4 Rosemary beetle (Chrysolina americana) feeding on lavender (Lavendula
angustifolia)..........................................................................................................125
4.3.4.1 Rosemary beetle..................................................................................125
4.3.4.2 Rosemary beetle gut bacteria.............................................................127
4.3.4.3 Antibiotic susceptibility testing...........................................................127
4.3.4.4 Lavender extract..................................................................................128
4.3.5 Death’s head hawkmoth (Acherontia atropos) feeding on potato (Solanum
tuberosum)...........................................................................................................132
4.3.5.1 Death’s head hawkmoth......................................................................132
4.3.5.2 Gut bacteria.........................................................................................133
4.3.5.3 Antibiotic susceptibility testing...........................................................134
4.3.5.4 Potato leaf extract ..............................................................................134
4.3.6 Beet armyworm (Spodoptera exigua) feeding on Madagascar periwinkle
(Catharanthus roseus)..........................................................................................137
4.3.6.1 Beet armyworm...................................................................................137
4.3.6.2 Catharanthus roseus............................................................................137
4.3.6.3 Antibiotic resistance in beet armyworm gut bacteria.........................139
4.3.6.4 Madagascar periwinkle extract...........................................................140
4.3.6.5 Extract fractionation............................................................................143
4.4 Discussion and Conclusions................................................................................149
4.4.1 Giant stick insect feeding on eucalyptus.....................................................149
4.4.1.1 Gut microbiota.....................................................................................149
4.4.1.2 Antibiotic resistance............................................................................150
4.4.1.3 Eucalyptus extract...............................................................................150
4.4.2 P. xylostella feeding on cabbage..................................................................151
4.4.2.1 Gut microbiota.....................................................................................151
4.4.2.2 Cabbage extract...................................................................................151
4.4.3 Cinnabar moth feeding on ragwort.............................................................152
4.4.3.1 Gut microbiota.....................................................................................152
4.4.3.2 Ragwort extract...................................................................................152
4.4.4 Rosemary beetle feeding on lavender........................................................153
4.4.4.1 Gut microbiota.....................................................................................153
4.4.4.2 Antibiotic resistance............................................................................153