The effect of flavonoids on the in vitro activity of antibiotics against Staphylococcus aureus

184 Pages·2013·13.84 MB·English

The effect of flavonoids on the in vitro activity of
antibiotics against St aphylococcus aureus

By

Tiza Lucy Ng’uni

Thesis submitted in Fulfillment of the Requirements for the Degree
Magister Scientiae

MSc

Medical Biosciences
Faculty of Natural Sciences
University of the Western Cape

Supervisor: Professor BC Fielding
Department of Medical Biosciences
University of the Western Cape

Co-Supervisor: Professor J Klaasen
Department of Medical Biosciences
University of the Western Cape

November 2012
KEYWORDS
Staphylococcus aureus  

Methicillin-resistant Staphylococcus aureus (MRSA)

Multidrug resistance

Flavonoids
Antimicrobial susceptibility testing
Combination studies
Synergistic ratios
Efflux pumps
Efflux pump genes
Polymerase Chain Reaction (PCR)

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ABSTRACT

Staphylococcus aureus is a Gram-positive coccus belonging to the Stapylococcaeae

family. S. aureus causes a wide range of infections that range from skin infections to life-

threatening infections such as pneumonia and endocarditis and is the major cause of hospital

and community-acquired infections. Despite antibiotics being available for the treatment of S.
aureus infections, resistance to a number of antibiotics has developed over the years due to
their improper and continuous use. S. aureus develops resistance to various drugs via
different mechanisms, one of which is the extrusion of the antibiotics through efflux pumps
that play a role in its acquisition of multidrug resistance. The ability of methicillin-resistant S.
aureus to develop resistance to a variety of antibiotics is causing global concern as treatment
options are being limited. Various antimicrobial studies carried out on purified plant-based
flavonoids have shown that flavonoids enhance the antibacterial effect of antibiotics.
This study analysed antibacterial effects of the antibiotics; tetracycline, ampicillin,
methicillin and vancomycin and three flavonoids; chrysin, naringenin and 7-hydroxyflavone,
against methicillin-sensitive ATCC 25923 (MSSA) and methicillin-resistant ATCC 33591
(MRSA) S. aureus strains, using the Kirby-Bauer disk diffusion and microtitre microdilution
assays. In the Kirby- Bauer assay, the antibiotics demonstrated inhibitory effects on the
growth of MSSA ATCC 25923. However MRSA ATCC 33591 was only susceptible to
vancomycin, with minimal inhibition zones observed with ampicillin. The flavonoids did not
enhance or reduce the antibacterial activities of the antibiotics as the zones of inhibition sizes
remained unchanged in the combination studies. Microtitre assay results revealed that
naringenin enhanced the antibacterial activities of the antibiotics tetracycline and ampicillin,
against MSSA ATCC 25923 and MRSA 33591. This was evident as calculated synergistic
ratios by the Abbot formula showed that naringenin had an additive effect. The presence of
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the efflux pump genes in MSSA ATCC 25923 and MRSA ATCC 33591 was compared using
polymerase chain reaction (PCR). The mepA and gyrA genes were identified in both strains

whereas sepA was identified in MRSA ATCC 33591. The presence of efflux pump genes in

both MSSA ATCC 25923 and MRSA ATCC  33591 also confirmed that the presence or
absence of the genes may contribute to antib iotic resistance. The presence of sepA in the
MRSA and not the MSSA confirmed that this gene plays a role in conferring drug resistance.

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DECLARATION

I, Tiza Lucy Ng’uni, declare that the thesis “The effect of flavonoids on the in vitro activity

of antibiotics against Staphylococcus aureu  s” is my own work, that it has not been
submitted for any degree or examination in any other university, and that all the sources I
have used or quoted have been indicated and acknowledged by complete references.

Tiza Lucy Ng’uni

Date............................................

Signed.........................................

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DEDICATION

This thesis is dedicated to my dad, Brig. Gen . Skinner Ng’uni, who passed away on 11th
May, 2012. Words cannot describe the pain  losing you has caused. Even though you are

gone, you will always be loved and always be in our hearts. May Your Soul Rest In Eternal
Peace.

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ACKNOWLEDGEMENTS

 I would firstly like to thank my God, for through Him all things are possible. I would

not have been able to do all this without Him.

 I would like to thank my supervisor, Professor BC Fielding. Thank you so much for
believing in me, supporting, encouraging and guiding me. You always go out of your
way to ensure that your students are taken care of and for that I am truly grateful. You
are the best supervisor one can ever ask for.
 I would also like to thank my co-supervisor, Professor JA Klaasen. Thank you for
your encouragement and guidance. I truly appreciate the time and effort you put into
helping me.
 To my virology lab colleagues, thank you for always being there and offering helping
hands each time I needed you. I will be forever grateful.
 To my family, my mum Mrs Catherine Ng’uni, baby girl and siblings. No words
can describe how much your support, love, encouragement, comfort and time means
to me. Mum, you and dad contributed to making me the person that I am today. I love
you all so much and I am truly grateful. I would also like to thank my friends, near
and far, for all your support.
 I would like to thank the National Research Foundation (NRF) and the
Department of Medical Biosciences, for the financial support and working space.

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TABLE OF CONTENTS
TITLE PAGE   i

KEYWORDS ii

ABSTRACT iii

DECLARATION v
DEDICATION vi
ACKNOWLEDGEMENTS vii
TABLE OF CONTENTS viii
LIST OF ABBREVIATIONS xiii
LIST OF FIGURES xvii
LIST OF TABLES xix
LIST OF APPENDICES xxi
CHAPTER ONE: Literature Review
1.1. Introduction 1
1.1.1. Infection 1
1.1.2. Diagnosis and identification 2
1.2. Virulence factors 4
1.2.1. Toxins 4
1.2.1.1. Superantigens 5
1.2.1.2. Exfoliative toxins 5
1.2.1.3. Other toxins 6
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1.2.2. Protein A 6
1.2.3. Role of staphyloxanthin in virulence   7

1.3. Treatment 8

1.4. Methicillin- resistant Staphylococcus aureus (MRSA) 11

1.4.1. Global prevalence and incidence of MRSA 12
1.4.2. Hospital-acquired MRSA (HA-MRSA) 14
1.4.3. Community-acquired MRSA 15
1.4.4. Livestock-associated MRSA (LA-MRSA) 16
1.4.5. Antimicrobial resistance of MRSA 18
1.4.5.1. Mechanism of antibiotic resistance 19
1.4.5.2. Methicillin resistance 22
1.4.5.3. β-lactam resistance 22
1.4.5.4. Tetracycline resistance 23
1.4.5.5. Aminoglycoside resistance 24
1.4.5.6. Quinolone resistance 25
1.4.5.7. Vancomycin resistance 26
1.5. Prevention and control 28
1.6. Efflux pumps and efflux pump genes 29
1.6.1. Types of efflux pumps 30
1.6.1.1. Primary transporters 30
1.6.1.2. Secondary transporters 31
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1.6.2. Staphylococcus aureus efflux pumps and efflux pump genes 32
1.6.3. Role of MDR pumps in antibiotic drug re sistance 33

1.6.4. Efflux pump inhibitors 34

1.7. Antimicrobial activities of flavonoids 35

1.8. Aims of the study 39
1.9. References 40
CHAPTER TWO: Effects of flavonoids on the in vitro antibacterial activities of
antibiotics against methicillin-sensitive and methicillin-resistant Staphylococcus aureus
2.1. Abstract 72
2.2. Introduction 73
2.3. Materials and Methods 76
2.3.1. Bacterial strains and growth conditions 76
2.3.2. Antibiotics and flavonoids 77
2.3.3. Antimicrobial susceptibility testing 77
2.3.3.1. Disk diffusion susceptibility test (Kirby-Bauer assay) 77
2.3.3.2. Microdilution susceptibility test (Microtitre assay) 79
2.3.4. Data analysis 81
2.4. Results 82
2.4.1. Antimicrobial susceptibility testing 82
2.4.1.1. Disk diffusion susceptibility test (Kirby-Bauer assay) 82
2.4.1.2. Microdilution susceptibility test (Microtitre assay) 85
2.5. Discussion 95
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