The Antibacterial Activity of Tea Infusions and Their Effect Against The Hospital Pathogen ...

258 Pages·2013·27.02 MB·English

The Antibacterial Activity of Tea
Infusions and Their Effect Against The
Hospital Pathogen Clostridium difficile

A thesis submitted for the degree of

Doctor of Philosophy (Ph.D)

by

William Francis McCully, MPharm

School of Pharmacy and Pharmaceutical Sciences
Cardiff University

June 2013
DECLARATION

Declaration
This work has not been submitted in substance for any other degree or award at this or any other
university or place of learning, nor is being submitted concurrently in candidature for any
degree or other award.

Signed ………………………………………… (candidate) Date 23/10/2013

STATEMENT 1

This thesis is being submitted in partial fulfillment of the requirements for the degree of PhD

Signed ………………………………………… (candidate) Date 23/10/2013

STATEMENT 2

This thesis is the result of my own independent work/investigation, except where otherwise
stated. Other sources are acknowledged by explicit references. The views expressed are my
own.

Signed ………………………………………… (candidate) Date 23/10/2013

STATEMENT 3

I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-
library loan, and for the title and summary to be made available to outside organisations.

Signed ………………………………………… (candidate) Date 23/10/2013

  i
ACKNOWLEDGMENTS

Acknowledgments
I must give the greatest thanks to my supervisors Professor Les Baillie, Dr Alex White
and Dr Natasha de Vere for all their insight, guidance and friendship during my time as
a post graduate student. I would like to pay particular thanks to Les for his support on a
daily basis.
I must also thank Dr Mandy Wootton for facilitating the work at the Anaerobic
reference Unit, UHW and Dr Jon Savage (Brighton University) for his assistance with
the electron microscopy.
Particular thanks goes to Dr Svetlana Ignatova and Dr Peter Hewitson (Brunel
University) for their advice and assistance with the use of HPLC and HPCCC.
I also must thank the Knowledge Economy Skills Scholarships (KESS) group for
funding this project.
I would also like to thank the members of the microbiology group for their support,
advice and most importantly their witty (or not so witty) banter over the last 3 years.
Finally I would like to thank my mother, partner and the rest of my family and friends
for their continued support and encouragement in everything I do.

  ii
ACKNOWLEDGMENTS

“Drinking a daily cup of tea will surely starve the apothecary“
- Old Chinese Proverb

  iii
SUMMARY

Summary
Clostridium difficile is one of the UK’s most common hospital acquired infections and
there is anecdotal evidence to suggest that the bacteria are sensitive to the antibacterial
properties of tea. Surprisingly, little research has been undertaken to characterise the
inhibitory activity of aqueous tea infusions that are representative of traditional drinking
habits. The antibacterial properties of tea are thought to be due to a group of
polyphenols called catechins. However, their contribution to the inhibitory activity of
tea infusions and their mechanism of action is still subject to debate.
An antimicrobial assay, developed using Staphylococcus aureus as a model organism,
was used to determine the antibacterial activity of a range of tea infusions against 75
clinical isolates of C. difficile that represented all the major strain ribotypes over 11
years.
Green teas demonstrated more potent antibacterial activity than black teas and their
activity was positively correlated with antioxidant power, hydrogen peroxide production,
and catechin content. Furthermore, the country of origin of the tea affected the catechin
content and subsequent antimicrobial activity of the infusion.
Detailed chemical analysis using high performance liquid chromatography and counter
current chromatography suggests that the antibacterial activity of tea is probably the
result of synergistic interactions between a number of catechins rather than the activity
of an individual compound. With regards to the mode of action by which tea inhibits C.
difficile, electron microscopy studies of the bacterium treated with green tea revealed
distinct changes to the outer cell structures of the bacteria. These changes were
indicative of cell membrane blebbing, thus supporting the theory that tea compounds
interact with the bacterial membrane and/or cell wall.
Overall, this investigation concluded that tea infusions have inhibitory activity against C.
difficile in vitro and may be useful in the treatment or prevention of C. difficile
infections in vivo.

  iv
ABBREVIATIONS

Abbreviations
AlCl Aluminium chloride
3
ANOVA Analysis of variance
ATP Adenosine triphosphate
BC-x Black commercial tea (x = associated tea sample number)
BHI Brain heart infusion
BP-x Black single plantation tea (x = associated tea sample number)
C Catechin
CDI C. difficile infection
CDT Binary toxin
CE Catechin equivalent
CFU Colony forming units
CI Confidence interval
CLSI Clinical and Laboratory Standards Institute
CO Carbon dioxide
2
Cu Copper
Da Dalton
diH O Deionised water
2
DNA Deoxyribonucleic acid
EC Epicatechin
ECG Epicatechin gallate
EGC Epigallocatechin
EGCG Epigallocatechin gallate
FDA Food and Drug Administration
Fe Iron
FIC Fractional Inhibitory Concentration
FOX Ferrous oxidation-xylenol orange
FRAP Ferric reducing antioxidant power
g gram
× g Gravitational force (Relative centrifugal force)
GA Gallic acid
GAE Gallic acid equivalent

  v
ABBREVIATIONS

GC Gallocatechin
GC-x Green commercial tea (x = associated tea sample number)
GCG Gallocatechin gallate
GP-x Green single plantation tea (x = associated tea sample number)
GTP Guanosine-5'-triphosphate
h hour
H Hydrogen
2
H O Hydrogen peroxide
2 2
HCl Hydrochloric acid
HPCCC High performance counter current chromatography
HPLC High performance liquid chromatography
L litre
LB Lysogeny broth
M Molar
Md Median
MH Mueller-Hinton
MIC Minimum inhibitory concentration
min minute
mL millilitre
MRSA Methicillin-resistant Staphylococcus aureus
n Number of replicates
N. Ireland Northern Ireland
N Nitrogen
2
NADPH Nicotinamide adenine dinucleotide phosphate
NaOH Sodium hydroxide
NB Nutrient Broth
NCIMB National Collection of Industrial, Food and Marine Bacteria
NCTC National Collection of Type Cultures
nm nanometre
NMR Nuclear magnetic resonance
O Oxygen
2
°C Degrees Celsius
OD Optical density

  vi
ABBREVIATIONS

p Value of significance
PBS Phosphate buffered saline
PCR Polymerase chain reaction
ppb Parts per billion
Q-Q Quantile-quantile
r r-value (Pearson product-moment correlation coefficient)
r2 Coefficient of determination
RNA Ribonucleic acid
SD Standard deviation
SE Standard error of the mean
sec second
SEM Scanning electron microscope
t t-test value
TcdA Toxin A
TcdB Toxin B
TE Trolox® equivalent
TEM Transmission electron microscope
TFC Total flavonoid content
TPC Total polyphenol content
TPTZ 2,4,6-tris(2-pyridyl)-1,3,5-triazine
TSA Tryptone soya agar
TSB Tryptone soya broth
U Mann-Whitney U test value
UHW University Hospital of Wales
UK United Kingdom
USA United States of America
UV Ultraviolet
v/v Volume per volume
w/v Weight per volume
w/w Weight per weight
WR Working reagent
z z-score
η2 eta squared (ratio of variance)

  vii
CONTENTS

Contents
DECLARATION
  I

ACKNOWLEDGMENTS
  II

SUMMARY
  IV

ABBREVIATIONS
  V

CONTENTS
  VIII

1.
 GENERAL
 INTRODUCTION
  2

1.1
 Natural
 Therapeutics
 Derived
 from
 Plants
  2

1.2
 Camellia
 sinensis
  4

1.3.
 Tea
 Leaf
 Processing
  8

1.3.1
 Withering
 or
 Wilting
  8

1.3.2
 Leaf
 Bruising
  10

1.3.3
 Oxidation
 (Fermentation)
  10

1.3.4
 Drying
  10

1.4
 Chemical
 Composition
 of
 Tea
 Leaves
  10

1.4.1
 Polyphenols
  11

1.4.1.1
 Flavonoids
  12

1.4.1.2
 Catechins
  14

1.4.1.3
 Flavonols
  16

1.4.1.4
 Theaflavins,
 Thearubigins
 and
 Theasinensins
  16

1.4.2
 Chlorophylls
 and
 Carotenoids
  19

1.4.3
 Methylxanthines
  21

1.5
 Environmental
 Factors
 Affecting
 the
 Polyphenol
 Content
 of
 C.
 sinensis
  22

1.5.1
 Seasonal
 Variation
  22

1.5.2
 Position
 of
 Tea
 Leaves
  22

1.5.3
 Light
  23

1.5.4
 Soil
 Composition
  24

1.5.5
 Water
  24

1.5.6
 Cultivating
 Altitude
  24

1.6
 The
 Health
 Benefits
 of
 Tea
  25

1.6.1
 Cardiovascular
 Disease
  25

1.6.2
 Antiviral
 Properties
  27

1.6.3
 Anticancer
 Properties
  28

1.6.4
 Antibacterial
 Properties
  29

1.7
 Clostridium
 difficile
  31

1.7.1
 C.
 difficile
 Toxins
  36

1.7.2
 Treatment
 of
 C.
 difficile
 Infection
 (CDI)
  38

1.7.2.1
 Antibiotics
  38

1.7.2.2
 Probiotics
  38

1.7.2.3
 Faecal
 Infusion
  39

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CONTENTS

1.7.3
 Tea
 as
 a
 Possible
 Treatment
 for
 C.
 difficile
 Infection
  40

1.8
 Aim
 and
 Objectives
 of
 this
 Study
  42

1.8.1
 Thesis
 Aim
  42

1.8.2
 Thesis
 objectives
  42

2.
 GENERAL
 MATERIALS
 AND
 METHODS
  44

2.1
 Aim
 and
 Objectives
  44

2.2
 Materials
  44

2.2.1
 Chemicals
 and
 Reagents
  44

2.2.2
 Biological
 Culture
 Media
  44

2.2.2.1
 Agars
 and
 Broths
  44

2.2.2.2
 Diluents
  45

2.2.3
 Bacterial
 Cultures
  45

2.2.3.1
 Staphylococcus
 aureus
  45

2.2.3.2
 Clostridium
 difficile
  45

2.2.4
 Tea
 Samples
  47

2.3
 Methods
  50

2.3.1
 Tea
 Infusion
 Preparation
  50

2.3.2
 Streak
 Plate
 Method
 for
 Determining
 Culture
 Purity
  50

2.3.3
 Gram
 Staining
 of
 Bacterial
 Cells
  51

2.4
 Statistical
 Analysis
  51

2.4.1
 Independent
 Samples
 t-­‐test
  51

2.4.2
 Mann-­‐Whitney
 U
 Test
  52

2.4.3
 One-­‐way
 Analysis
 of
 Variance
 (ANOVA)
  53

2.4.4
 Kruskal
 Wallis
 Test
  53

2.4.5
 Pearson’s
 Correlation
  54

2.4.6
 Spearman’s
 Correlation
  54

3.
 CHEMICAL
 ANALYSIS
 OF
 TEA
 INFUSIONS
  56

3.1
 Chapter
 Introduction
  56

3.1.1
 Colourimetric
 Assays
  56

3.1.1.1
 Total
 Polyphenol
 Content
 (TPC)
 Assay
  56

3.1.1.2
 Total
 Flavonoid
 Content
 (TFC)
 Assay
  57

3.1.1.3
 Ferric
 Reducing
 Antioxidant
 Power
 (FRAP)
 Assay
  57

3.1.1.4
 Hydrogen
 peroxide
 production
  58

3.1.2
 Quantitative
 Analysis
 -­‐
 High
 Performance
 Liquid
 Chromatography
 (HPLC)
  59

3.2
 Chapter
 Aim
 and
 Objectives
  60

3.3
 Methods
  61

3.3.1
 Colourimetric
 Assays
  61

3.3.1.1
 Determining
 the
 Total
 Polyphenol
 Content
 (TPC)
 of
 Aqueous
 Tea
 Infusions
  61

3.3.1.2
 Determining
 the
 Total
 Flavonoid
 Content
 (TFC)
 of
 Aqueous
 Tea
 Infusions
  61

3.3.1.3
 Determining
 the
 Ferric
 Reducing/Antioxidant
 Power
 (FRAP)
 of
 Aqueous
 Tea
 Infusions

  62

3.3.1.3.1
 Preparation
 of
 FRAP
 Reagent
  62

3.3.1.3.2
 Determining
 the
 Antioxidant
 Power
 of
 Aqueous
 Tea
 Infusions
  62

3.3.1.4
 Determining
 the
 H O
 production
 of
 Aqueous
 Tea
 Infusions
 using
 the
 FOX
 assay
  63

2 2
3.3.1.4.1
 Preparation
 of
 working
 reagent
  63

3.3.1.4.2
 Determining
 the
 H O
 production
 of
 Aqueous
 Tea
 Infusions
  63

2 2
3.3.2
 HPLC
 Analysis
 of
 Aqueous
 Tea
 Extracts
  63

3.3.2.1
 Preparation
 of
 tea
 extracts
 and
 standards
  63

  ix