Honey as an antimicrobial agent against multi-drug resistant Gram negative bacterial rods

270 Pages·2011·17.54 MB·English

Honey as an antimicrobial agent against

multi-drug resistant Gram negative
bacterial rods
By
Rahma Ali Saleh Al-Maaini

This dissertation is submitted in partial fulfilment of the
degree of MPhil in Biomedical Sciences
(Microbiology)
School of Health Sciences
University of Wales Institute Cardiff, UK
May 2011

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Contents

Title I
Contents II
Declaration XI
Dedication XII
Acknowledgments XIII
Poster Presentation XIV
Index of Tables XV
Index of Figures XIX
Abbreviations XXVI
Abstract XXX
Chapter 1: Introduction 1
1.1 Antimicrobial Resistance: 2
1.2 Extended Spectrum Beta-Lactamases (ESBLs): 3
1.2.1 Classification of ESBL: 4
1.2.1.1 Functional Classification: 4
1.2.1.2 Molecular Classification: 4
1.2.2 Types of ESBL: 5
1.2.2.1 TEM-type ESBLs (class A) 5
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1.2.2.2 SHV-type ESBLs (class A)
1.2.2.3 CTX-M type ESBLs (class A): 6
1.2.2.4 OXA-type ESBLs (class D) 7
1.2.2. 5 AmpC-type ESBLs (class C) 7
1.2.2.6 Carbapenemases (class A, B, D) 8
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1.2.3 Emergence of ESBL: 9
1.2.4 Risk factor for ESBL: 9
1.2.5 Treatment of ESBLs: 10
1.3 Acinetobacter: 10
1.3.1 Taxonomy and Historical Features of Acinetobacter: 11
1.3.2 Laboratory Diagnosis: 12
1.3.3 Clinical features of Acinetobacter infections: 12
1.3.4 Pathogenesis of Acinetobacter infection: 14
1.3.4.1 The production of exopolysaccharide: 15
1.3.4.2 Quorum-sensing: 15
1.3.4.3 The property of adhesion to human epithelial cells via 1 5
the capsule or fimbriae.
1.3.4.4 Surface and mitochondrial porins: 16
1.3.5 Emergence of Resistance: 17
1.3.6 Carbapenem action on Acinetobacter: 18
1.3.7 Mechanisms of Carbapenem Resistance: 18
1.3.8 Acinetobatcer Treatment with Honey: 20
1.4 Alternative Antimicrobial Therapies: 21
1.4.1 Ancient Use of Honey as a Medicine: 22
1.4.2 Honey as a Modern Medicine: 22
1.4.3 Antimicrobial Activity of Honey: 24
1.4.4 Chemical composition of honey: 26
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1.4.5 Factors Contributing Antibacterial Properties of Honey: 27
1.4.5.1 Osmotic effect: 28
1.4.5.2 Acidity 28
1.4.5.3 Hydrogen peroxide production 29
1.4.5.4 Non-peroxide Components 30
1.4.5.5 Antioxidant activity: 31
1.4.5 Manuka Honey: 34
1.4.6 Omani Honey: 36
1.5 Aims and Objectives: 39
Chapter 2: Materials & Methods: 40
2.1 Characterization of test organisms: 49
2.1.1 Confirmation of the identity of test organisms: 49
2.1.2 Antibiotics sensitivities: Extended Spectrum Beta- lactamases 49
test (ESBLs):
2.1.2.1 Inoculum preparation: 49
2.1.2.2 Disc application: 49
2.1.2.3 Screening test for ESBL: 50
2.1.2.4 Phenotypic confirmatory test for ESBL: 50
2.1.2.5 ESBL/AmpC* confirmation test: 50
2.2 Characterization of honey samples: 51
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2.2.1 Honey sample collection: 51
2.2.2 Bioassay of antibacterial activity: 51
2.2.2.1 Phenol standards preparation for bioassay: 52
2.2.2.3 Inoculum preparation: 52
2.2.2.4 Plate preparation: 52
2.2.2.5 Sample preparation: 53
2.2.2.6 Samples and standards application: 53
2.2.2.7 Zone measurement: 54
2.2.2.8 Calculation of antibacterial activity of honey: 54
2.2.3 Determination of pH: 55
2.2.4 Sugar and water content: 55
2.2.5 Hydroxymethylfurfural (HMF) concentration: 56
2.2.6 Protein content: 56
2.2.7 Colour: 58
2.2.8 Pollen analysis: 58
2.2.9 Total phenolic content: 59
2.2.9.1 Reagent/standard preparation: 59
2.2.9.2 Assay method: 60
2.2.10 Free radical activity of honey: 61
2.2.10.1 Reagent / Standard Preparations 61
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2.2.10.2 Assay procedure: 62
2.3 Determination of antibacterial activity of honey samples against test 62
cultures:
2.3.1 Minimum Inhibitory Concentration (MIC) method: 62
2.3.1.1 Agar incorporation method: 62
2.3.1.1.1 Honey selection: 62
2.3.1.1.2 Inoculum preparation: 63
2.3.1.1.3 Plates preparation: 63
2.3.1.1.4 Honey preparation: 63
2.3.1.1.5 Plates reading (MIC determination): 64
2.3.1.2 Broth dilution method:
2.3.1.2.1 Honey selection: 65
2.3.1.2.2 Honey dilution: 65
2.3.2.1.3 Microtitre plate inoculation: 65
2.3.2.1.4 Visual inspection of MIC: 66
2.3.2.1.5 Spectrophotometric determination of MIC: 66
2.3.2 Minimum Bactericidal concentration (MBC) method: 66
2.4 Time Kill Curve Assay: 67
2.4.1 Strain selection: 67
2.4.2 Time-kill curve: 67
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2.5 Effect of honey on bacterial structure: 68
2.5.1 Scanning Electron Microscopy (SEM): 68
2.5.1.1 Bacterial Selection for SEM 68
2.5.1.2 Preparation of cells in the exponential phase of growth: 69
2.5.1.3 Preparation of cells for scanning electron microscopy: 69
2.5.2 Transmission Electron Microscopy (TEM): 70
2.5.2.1 Bacterial Selection for TEM 70
2.5.2.2 Preparation of cells for transmission electron 71
microscopy:
2.5.2.3 Pallet trimming and sectioning: 71
2.5.2.4 Staining of thin sections 71
2.6 Effect of honey on bacterial proteins: 72
2.6.1 Two Dimensional Gel Electrophoresis: 72
2.6.1.1 Buffers preparation: 73
2.6.1.2 Cell preparation: 74
2.6.1.3 Protein determination of Acinetobacter extracts for each 74
extract:
2.6.1.4 Rehydration and sample application: 76
2.6.1.5 Rehydration in the PROTEAN® IEF Focusing Tray: 76
2.6.1.6 Equilibration and SDS-PAGE: 77
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2.6.1.7 Staining and gel visualising: 77
2.7 Statistical analysis of the data: 78
Chapter 3: Results 79
3.1 Confirmations of the identity and antibiotics sensitivities of test 80
organisms:
3.2 Characterization of honey samples: 88
3.2.1 Determination of antibacterial activity: 88
3.2.2 Chemical & physical analysis of honey samples 91
3.2.2.1 Pollen analysis 94
3.2.2.2 Antioxidant activity assay: 96
3.3 Determination of antibacterial activity of honey samples against test 97
cultures:
3.3.1 Minimum Inhibitory Concentration (MIC) and Minimum 97
Bactericidal Concentration (MBC) of manuka honey:
3.3.1.1 Agar incorporation method: 97
3.3.1.2 Broth dilution method: 99
3.3.2 Sensitivity of MDR and ESBLs to Omani honey: 107
3.4 Time Kill Curves 115
3.4.1 Inhibition of test organisms by manuka honey monitored by 115
optical density:
3.4.2 Inhibition of test organisms by manuka honey monitored by total 119
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viable count
3.4.2.1 Acinetobacter spp 120
3 3.4.2.2 E.coli 122
3.4.2.3 Klebsiella 124
3.4.2.4 Citrobacter 126
3.4.2.5 Enterobacter 128
3.4.2.6 Serratia 130
3.5 Effect of honey on bacterial structure: 134
3.5.1 Growth Curves: 134
3.5.2 Scanning Electron Microscopy (SEM): 138
3.5.2.1 SEM of Acinetobacter 138
3.5.2.2 SEM of E.coli 148
3.5.2.3 SEM of Klebsiella 151
3.5.2.4 SEM of Enterobacter 160
3.5.2.5 SEM of Citrobacter 165
3.5.2.6 SEM of Serratia 170
3.5.2 Transmission Electron Microscopy (TEM): 174
3.5.2.1 TEM for Acinetobacter: 174
3.5.2.2. TEM for E.coli: 179
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3.6 Effect of honey on bacterial proteins: 184
3.6.1 Two Dimensional Gel Electrophoresis: 184
Chapter 4: Discussion 186
4.1 Antibacterial activity of honey samples: 187
4.2. Chemical & physical analysis of honey samples 189
4.3 Effect of honey samples against test cultures: 193
4.4 Inhibition of test organisms by manuka honey using time kill curve assay 201
4.5 Effect of honey on bacterial structure: 202
4.6 Further investigations: 213
Chapter 5: References 215

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