The Effects of Garlic on the Susceptibility of MRSA to p-lactam Antibiotics By Jonathan Wood A ...

234 Pages·2013·16.32 MB·English

C U L T E C H
Cardiff
UNIVERSITY
PRIFYSGOL
CArRDV|9
BIOSHOALriY PRODUCTS
The Effects of Garlic on the Susceptibility of
MRSA to p-lactam Antibiotics
By
Jonathan Wood
A thesis presented for the degree of
Doctor of Philosophy
At
The School of Biosciences,
Cardiff University
May 2009
UMI Number: U584400
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Acknowledgements
I would not have got this far were it not for a tremendous amount of support from a
number of people.
Cultech Ltd and Obsidian Research Ltd
Thanks to everyone at Cultech and Obsidian for the warm welcome they provided and
for the many laughs along the way. Specifically I would like to thank my supervisors
Dr. Sue Plummer and Dr. Iveta Garaiova whose support, advice and friendship have
been much appreciated. Additionally I would like to thank Mark Weaver for his
constant enthusiasm and encouragement.
Cardiff University
First and foremost I would like to thank Prof. David Lloyd, whose extensive
knowledge is an inspiration, and whose advice, encouragement and support has been
invaluable.
Thanks to Carsten Muller, for his support and friendship throughout my time at
Cardiff, not just over the past four years.
Thanks also to Dr. Martin Day who has provided a huge amount of support and
advice.
I would also like to thank Dr. Hefin Jones - thanks for your friendship, advice and
encouragement over the past few years.
Thanks to all my fellow PhD adventurers in the Microbiology department and my
adopted BEPG. Thanks for the tea, cakes, parties, pints and good times! In particular I
would like to thank David, Geoff, Wendy and Phil who’ve probably put up with me
more than the others - sorry ‘bout that!
I must also thank my friends and family, particularly Mum, Dad, Chris and Debs -
this wouldn’t have been possible without your love, support and belief.
And finally, I must thank my wonderful girlfriend Bozena - dzi^kuj? Ci bardzo za
wszystko. Nie zrobilbym tego bez Ciebie.
Publications and Presentations
Publications
Wood, J., Plummer, S., Garaiova, I., Muller, C. and Lloyd, D. Review: The
antibacterial effects of garlic and its sulphur containing compounds. IN
PREPARATION
Wood, J., Cottrell, S.L., Plummer, S., Garaiova, I., Hann, A.C., Muller, C. and Lloyd,
D. Bacteriostatic and inhibitory effects of garlic against Escherichia coli,
Staphylococcus aureus and Lactobacillus casei. IN PREPARATION
Wood, J., Plummer, S., Garaiova, I., Muller, C. and Lloyd, D. In vitro reduced
susceptibility of S. aureus to garlic through repeated sub-culture in sub-inhibitory
concentrations. IN PREPARATION
Wood, J., Plummer, S., Garaiova, I., Muller, C. and Lloyd, D. The effects of garlic on
the growth of MRSA and Staphylococcus aureus. IN PREPARATION
External poster presentations
Wood, J., Garaiova, I., Plummer, S., Muller, C., Day, M. and Lloyd, D. The effect of
garlic on the sensitivity of meticillin-resistant Staphylococcus aureus to oxacillin.
Society for General Microbiology (SGM) 162nd Meeting - Edinburgh International
Conference Centre, UK, 31st March - 3rd April 2008
Norville, P., Wood, J., Garaiova, I., Plummer, S., Mtiller, C., Day, M. and Lloyd, D.
Different responses to combinations of green tea and oxacillin exhibited by
Staphylococcus aureus small colony variants and their parents. 13 ♦Vi International
Symposium on Staphylococci and Staphylococcal Infections (ISSSI) - Caims
Convention Centre, Australia, 7th - 10th September 2008
Wood, J., Garaiova, I., Plummer, S., Muller, C., Day, M. and Lloyd, D. In vitro
production of antimicrobial resistance in a methicillin-sensitive strain of
Staphylococcus aureus. 13th International Symposium on Staphylococci and
Staphylococcal Infections (ISSSI) - Caims Convention Centre, Australia, 7th - 10th
September 2008
Abstract
Abstract
It is well recognised that resistance to antibiotics is a major global health problem
which is increasing in significance year on year. Many authorities believe it to be the
biggest healthcare issue of the 21st century. In particular, methicillin-resistant
Staphylococcus aureus (MRSA) is a major clinical problem, particularly in the UK.
Plant-derived products, and particularly garlic, have been recognised as having
antimicrobial activity for centuries. This traditional use has now been substantiated by
credible science. Recent studies have begun to investigate whether there is any
synergism between clinically-employed antimicrobials and plant-derived products,
which could, in theory, make use of our currently redundant antibiotics
In this study sub-MIC concentrations of garlic (MICs 1-2 mg mL*1) were shown to
potentiate oxacillin susceptibility (MIC < 4 pg mL"1) against MRSA, and increase the
sensitivity of MRSA to penicillin. Results suggest that increasing antibiotic
susceptibility was as a result of the cumulative effects of the garlic and antibiotic
rather than interaction between the two. It therefore appears that garlic does not
exhibit the same mechanism of action as oxacillin or penicillin. Low concentrations of
garlic (<0.5 mg mL"1), however, decreased the susceptibilities of MRSA and S.
aureus to oxacillin or penicillin. Garlic caused thickening of the primary
Staphylococcal cell wall, but did not damage the cell wall integrity.
This is the first study to show in vitro decreasing sensitivity to garlic through repeated
sub-culture in sub-inhibitory concentrations (0.5x MIC). Such isolates exhibited
morphological changes but did not differ from control cultures in terms of growth
dynamics. Acquired resistance to garlic has not previously been reported, suggesting
these data are worthy of further investigation.
The present study lends further support to the continued investigation into
potentiation of antibiotic susceptibility in antibiotic-resistant organisms by
phytochemicals. The results presented here do however suggest that these interactions
may be more complex than anticipated and that low concentrations of phytochemicals
could potentially exacerbate the problem of antibiotic-resistance.
Page
Declaration
Acknowledgements i
Publications and Presentations ii
Summary iii
Table of Contents iv
Figure Legend ix
Table Legend xii
Abbreviations xiii
1 General Introduction
1.1 Staphylococcus aureus and MRSA 1
1.1.1 Morphology 1
1.1.2 Methicillin-resistant Staphylococcus aureus (MRSA) 2
1.2 Antibiotic resistance 3
1.2.1 Antibiotic resistance mechanisms and P-lactam resistance in 3
MRSA
1.2.2 Heterogeneous methicillin resistance in MRSA 4
1.2.3 Antibiotic resistance: a global health problem 4
1.2.4 Combating antibiotic resistance 8
1.3 Garlic 10
1.3.1 Botany and classification 10
1.3.2 The historical uses of garlic 11
1.3.4 The sulphur chemistry of garlic 12
1.3.4.1 Allicin 14
1.3.5 Aqueous preparations of freeze-dried garlic powder (FGP) 15
1.3.6 Reported health promoting benefits of garlic 16
1.3.7 Antiprotozoal effects of garlic 16
1.3.8 Anti fungal effects of garlic 17
1.3.9 Antiviral effects of garlic 19
1.3.10 Antibacterial effects of garlic 19
1.3.10.1 Antibacterial effects of allicin (or garlic) 19
1.3.10.2 Antibacterial effects of diallyl sulphides 22
1.3.10.3 Antibacterial effects of ajoene and allyl alcohol 22
1.4 Increases in turbidity as a measure of growth of organisms in 23
liquid cultures
2 General Materials and Methods
2.1 Strains and isolates 25
2.2 Media 26
2.3 Preparation of aqueous garlic solutions 26
2.4 Preparation of oxacillin and penicillin solutions 26
2.5 Inocula (based upon CLSI guidelines; CLSI2007) 27
iv
2.6 Determination of viable counts (plate counts) 27
2.7 Bioscreen C - Determinations of MICs and chequerboard 28
assays
2.7.1 Bioscreen C growth analyser parameters and set-up 28
2.7.2 Determinations of MICs using a Bioscreen 29
2.7.3 Chequerboard assays using a Bioscreen 29
2.7.4 Determinations of growth dynamics from Bioscreeen growth 30
curves
2.8 Visualisations of cellular morphologies by electron 30
microscopy
2.8.1 Further sample preparation for scanning electron microscopy 31
(SEM)
2.8.2 Further sample preparation for transmission electron 31
microscopy
The Effects of Garlic on the Growth of MRSA and S. aureus
3.1 Introduction 32
3.1.1 The problem of antibiotic resistance 32
3.1.2 The rapid progression of antibiotic resistance 33
3.1.3 Alternative therapies to traditional antibiotics 34
3.1.4 Antimicrobials derived from plants 35
3.1.5 The present study 37
3.1.6 Aims 38
3.1.7 Hypotheses 38
3.2 Materials and Methods 39
3.2.1 Effect of garlic on the growth of MRSA and S. aureus 39
3.2.2 Effect of garlic on the growth dynamics of MRSA, S. aureus 39
and E. coli
3.2.3 Effect of garlic on the osmotic tolerance of MRSA and E. 39
coli
3.2.4 Effect of garlic on the cellular morphology of MRSA and S. 39
aureus determined using scanning electron microscopy
3.3 Results 40
3.4 Discussion 51
3.4.1 The susceptibility of MRSA and S. aureus to garlic 51
3.4.2 Effect of garlic on Staphylococcal growth dynamics 52
3.4.3 Effect of garlic on the osmotic tolerance of MRSA 53
3.4.4 Effect of garlic on EMRSA-16 55
3.5 Conclusions 56
The Effects of P-lactam Antibiotics on the Growth of MRSA and S. aureus
4.1 Introduction 57
4.1.1 The bacterial cell wall 57
4.1.2 P-lactam antibiotics 57
4.1.3 Mechanisms of p-lactam resistance 59
4.1.4 The use of oxacillin and penicillin in the present study 60
4.1.5 Aims 60
4.2 Materials and Methods 61
4.2.1 Detection of P-lactamase expression 61
4.2.2 Disc diffusion 61
V
4.2.3 Viability on oxacillin containing agar 61
4.2.4 Effect of oxacillin and penicillin on MRSA and S. aureus 62
growth dynamics
4.3 Results 63
4.4 Discussion 69
4.5 Conclusions 71
The Effects of Garlic on the p-lactam Susceptibility of MRSA and S. aureus
5.1 Introduction 72
5.1.1 Activity of antimicrobials derived from plants compared 72
with traditional antibiotics
5.1.2 Potentiation of antibiotic susceptibility in MRSA 73
5.1.3 Interactions between garlic compounds and antibiotics 76
5.1.4 Quantification of interactions between two antimicrobial 80
compounds
5.1.5 The present study 81
5.1.6 Aims 81
5.2 The effect of pre-treatment with garlic on the oxacillin 82
susceptibility of MRSA and S. aureus
5.2.1 Background 82
5.2.2 Aims 82
5.2.3 Hypotheses 82
5.2.4 Materials and methods 83
5.2.5 Results 84
5.2.6 Discussion 89
5.2.7 Conclusions 90
5.3 The effect of combinations of garlic and oxacillin against 91
MRSA and S. aureus
5.3.1 Background 91
5.3.2 Aims 91
5.3.3 Hypotheses 91
5.3.4 Materials and methods 92
5.3.5 Results 93
5.3.5.1 The effect of garlic and P-lactam antibiotics on 97
Staphylococcal growth dynamics
5.3.6 Discussion 100
5.3.7 Conclusions 101
5.4 The effects of low concentrations of garlic on the 102
susceptibility of MRSA and S. aureus to oxacillin
5.4.1 Background 102
5.4.2 Aims 102
5.4.3 Materials and methods 103
5.4.3.1 Effect of combinations of garlic and oxacillin (at small 103
increments) on MRSA and S. aureus
5.4.3.2 Transmission electron microscopy of S. aureus NCTC 6571 103
treated with 0.375 mg mL'1 garlic
5.4.4 Results 104
5.4.5 Discussion 110
5.5 Overall discussion 112
5.6 Conclusions 115
Induction of Antimicrobial Resistance in Oxacillin Susceptible S. aureus
6.1 Introduction 116
6.1.1 Bacterial resistance to garlic 116
6.1.2 Mechanisms of antibiotic resistance 117
6.1.3 Induction of non-p-lactamase mediated resistance to 119
penicillin
6.1.4 Aims 120
6.1.5 Hypotheses 121
6.2 Materials and methods 123
6.2.1 General method 123
6.2.2 Effect of treatment on growth dynamics 125
6.2.3 Visualisation of abnormal cellular morphologies by scanning 125
and transmission electron microscopy
6.2.4 Statistical analysis 126
6.3 Results 127
6.3.1 Colonial morphological changes 129
6.3.2 The effect of treatment on growth dynamics 130
6.3.3 Cellular morphological changes 132
6.3.4 Maintenance of resistance 133
6.4 Discussion 137
6.4.1 Induction of oxacillin resistance 137
6.4.2 Decreased garlic susceptibility in S. aureus NCTC 6571 139
6.5 Conclusions 141
S. aureus Ox-RI is a Phenotypic Mutant of S. aureus NCTC 6571:
Confirmation by RAPD Fingerprinting
7.1 Introduction 142
7.1.1 Use of RAPD fingerprinting in the present study 143
7.1.2 Aims 144
7.1.3 Hypothesis 144
7.2 Materials and methods 145
7.2.1 Identification by Gram morphology and substrate utilisation 145
7.2.2 Chelex® 100 DNA extraction method 145
7.2.3 RAPD method 145
7.2.4 Gel electrophoresis 146
7.2.5 Imaging 146
7.3 Results 147
7.4 Discussion 148
The Susceptibility of MRSA and S. aureus to Plant Antimicrobials Other
than Garlic
8.1 Introduction 149
8.1.1 Oregano - Origanum vulgare 149
8.1.2 Cinnamon - Cinnamomum zeylanicum (Synonym: 150
Cinnamomum verum)
8.1.3 Green tea (Camilla sinensis) 152
8.1.4 The present study 153
8.1.5 Aims 154
8.1.6 Hypotheses 154
8.2 Essential oils and antimicrobial powders 155
vii