TABLE OF CONTENTS
Title page - - - - - - - - - - I
Certification - - - - - - - - - - ii
Declaration - - - - - - - - - iii
Dedication - - - - - - - - - iv
Acknowledgements - - - - - - - - - - - - - - vi
Table of Contents - - - - - - - - - - - vii
List of Tables - - - - - - - ix
List of Figures - - - - - - - - - - - - xvi
List of Plates - - - - - - - - - - - - xviii
Abstract - - - - - - - - - - xix
CHAPTER ONE: INTRODUCTION
11Background - - - - - - - - 1
12 Importance of this research - - - - - - - - - - 3
13 The aim and objectives of the research - - - - - - - - - 4
CHAPTER TWO: LITERATURE REVIEW
21 The staphylococci - - - - - - - - 6
211 Important properties - - - - - - 6
212 Staphylococcus aureus genome - - - - - - - - - - - - - - - - 7
213 Staphylococcus aureus cell Wall - - - - - 9
22 Staphylococcus aureus virulence determinants - - - - 10
221 Exfoliative toxins - - - - - - - - 11
222 Adhesins - - - - - - - 12
223 Superantigenic exotoxins - - - - - - - - 13
224 Staphylococcus aureus enzymes - - - - - 14
225 Leukocidins - - - - - - 15
23 Staphylococcus aureus pathogenesis - - - - 15
23
1 Staphylococcus aureus infections - - - - - 18
24 Nasal carriage of Staphylococcus aureus - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 20
241 Staphylococcus aureus colonization among health-care workers - - - - 21
242 The environment as a source of infection - - - - - - 22
25 Coagulase negative staphylococci - - - - - 23
251 Biofilm formation in staphylococci - - - - - - - - 26
26 Emergence of antimicrobial resistance - - - - - - - 27
261 Genetics of antimicrobial resistance - - - - - - - - 28
262 Evolution of methicillin resistant S aureus (MRSA) - - - - - - - - - - 28
27 Antistaphylococcal agents - - - - - - - - 33
271 β-lactam antibiotics - - - - - - - 33
272 Penicillins - - - - - - - 33
273 Penicillinase-stable β-lactams - - - - - - - - - - - 36
274 Genetics of methicillin resistance - - - - 36
275 Macrolides, Lincosamides & Streptogramins - - - - 39
276 Fluoroquinolones - - - - - - - 40
277 Aminoglycosides - - - - - - - - 41
278 Tetracyclines - - - - - - - - - 41
279 Sulfonamides & Trimethoprim - - - - - - - - - - - 42
2710 Glycopeptides - - - - - - - - - - 43
2711 Linezolid - - - - - - - 44
2712 Daptomycin - - - - - - - - - - - 44
2713 Tigecycline - - - - - - 45
2714 Quinupristinâ€"dalfopristin - - - - - - - - - 45
28 Epidemiology of S aureus infections - - - - - - - - 50
29 Molecular typing methods for S aureus - - - - - - - 54
291 Antibiogram - - - - - - - - - - 56
292 Bacteriophage typing - - - - - - 56
293 Plasmid profile analysis - - - - - - - - - 57
294 Pulsed Field Gel electrophoresis (PFGE) - - - - - - - 58
295 Polymerase Chain Reaction (PCR) - - - - - 59
296 Real-Time Polymerase Chain Reaction - - - - - - - - 61
297 Coagulase gene typing - - - - - - - - - - 61
298 Staphylococcal protein A (spa) typing - - - - - - - 62
299 Staphylococcal Cassette Chromosome mec (SCCmec) typing - - - - - 63
2910 Multilocus Sequence Typing (MLST) - - - - - - - 64
210 Strategies for control of S aureus infections - - - - - - - 65
2101 Hand washing - - - - - - - - - 66
2102 Screening of staff - - - - - - - - 67
2103 Environmental cleaning - - - - - - - - - 68
CHAPTER THREE: MATERIALS AND METHODS
31 Materials - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 69
311 Bacterial isolates - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 69
312 Reference bacterial strains - - - - - - - - 69
313 Antimicrobial agents - - - - - - - - - - - - - - - - - - - - - - - - 70
314 Polymerase chain reaction (PCR) primers - - - - - - - - - - - - - - - - 70
315 Centers of study - - - - - - - - - - 70
32 Methods - - - - - - - - - - - - - - - - - 75
321 Collection of bacterial samples - - - - - - - 75
322 Patient demographics - - - - - - - 75
323 Maintenance of bacterial strains - - - - - - - - - - - - 75
33 Identification of bacterial isolates - - - - - - 76
331 Gram stain - - - - - - - 76
332 Catalase test - - - - - - 76
333 Coagulase test - - - - - - - - - - 76
334 Latex agglutination test - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 77
335 Identification with VITEK-2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 77
34 Antimicrobial susceptibility testing - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 79
341 Antibiotic susceptibility testing by disk diffusion technique - - - - - - - 79
342 Antimicrobial susceptibility testing with VITEK-2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 80
35 Molecular techniques used - - - - - - - - - - - - - - - - - - - - - - - - - - - - 81
351 DNA extraction for amplification - - - - - - - - - - - - - - - - - - - - - - - - 81
352 Polymerase chain reaction (PCR) - - - - - 81
353 Agarose Gel Electrophoresis - - - - - - - 81
36 Identification of S aureus by detection of nuc gene - - - - - - - - - - - - - - - - - - - - 82
37 Detection of antibiotic resistance genes - - - - - - - 83
371 Detection of the beta-lactamase gene (blaZ) gene - - - 83
372 Amplification of the mecA gene - - - - - 83
373 Characterization of SCCmec - - - - - - - 84
38 Accessory gene regulator (agr) typing - - - - - - - - 84
39 Characterization of virulence determinants - - - - - - - - - 84
391 Detection of capsular genotype - - - - - - 84
392 Staphylococcal toxin gene detection - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 85
393 Detection of lukS-PV and lukF-PV - - - - - - - 85
394 Quantitative detection of the PVL protein - - - - - - 86
3941 Extraction of LukS-PV - - - - - 86
3942 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) - - - - - 86
3943 Gel preparation - - - - - - - 87
3944 Electrophoresis - - - - - - - 87
3945 Western Blot - - - - - - - - 88
310 Genotyping of S aureus strains - - - - - - - 89
3101 Protein A (spa) gene amplification - - - - 89
311 Transformation experiments - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 90
3121 Making electrocompetent cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 91
3122 Transformation of S aureus RN4220 by electroporation - - - - - - - - - - - - - - - - - - - - - - - - 91
3123 Extraction of plasmid DNA - - - - - - - - 92
3124 Transformation of S aureus strains - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 92
313 Statistical analysis - - - - - - - - - - - - 93
CHAPTER FOUR: RESULTS
41 Identification of isolates used in this study - - - - - - - - - 94
42 Antimicrobial susceptibility profile of S aureus strains - - - - 94
43 Comparison of antimicrobial susceptibility profile of S aureus and coagulase negative
staphylococcal strains - - - - - - - - - - - - - - - 96
44 Identification of antibiotic resistance genes in S aureus strains - - - - - 97
45 Identification of virulence determinants in S aureus isolates - - 110
451 Identification of the capsular polysaccharides in S aureus isolates - - - 110
452 Prevalence of toxin genes in S aureus strains - - - - 110
453 Comparison of virulence determinants in S aureus isolates from clinical, carrier and
environmental sources - - - - - - - - - - - 110
454 Disease-association of PVL - - - - - - 111
455 Detection and quantification of LukS-PV protein in PVL-gene positive S aureus isolates
- - - - - - - - - - - - - 112
46 agr typing and relationship with virulence determinants - - - 112
47 Molecular diversity of S aureus based staphylococcal protein A (spa) typing - - - - 128
48 Characterization of MRSA strains - - - - - - - - 129
49 Transfer of plasmid-mediated antibiotic resistance to 2 tetracycline-suceptible S aureus
strains - - - - - - - - - - - - 137
CHAPTER FIVE: DISCUSSION
51 Identification and confirmation of S aureus strains - - - - - - 139
52 Antibiotic resistance in S aureus strains - - - - - - 140
521 Comparison of antimicrobial susceptibility pattern of S aureus with coagulase negative
staphylococci - - - - - - - - - 141
53 Detection of antibiotic resistance genes - - - - - - - 143
54 Identification of virulence determinants in S aureus isolates - - 146
541 l Identification of the capsular polysaccharides in S aureus isolates - - - - - - - 146
542 Staphylococcus aureus toxin genes - - - - 147
543 Prevalence of Panton Valentine Leukocidin (PVL) genes and the expression of its protein
in S aureus isolates - - - - - 148
55 Relationship agr groups and virulence determinants - - - - - - 151
56 Molecular diversity of S aureus isolates based on staphylococcal protein A (spa)
typing - - - - - - - - 153
57 Transfer of plasmid-mediated antibiotic resistance to Staphylococcus aureus strains156
CONCLUSION AND RECOMMENDATIONS - - - - - 158
CONTRIBUTIONS TO KNOWLEDGE - - - - - - - - 160
REFERENCES - - - - - - - 161
APPENDICES
LIST OF TABLES
Table 21: Examples of antimicrobial resistance determinants in S aureus - - - - - - - - 47
Table 31 PCR primers used in this study - - - - - - - - - - - - - - - - - - 73
Table 41 Distribution of staphylococcal isolates obtained from clinical, carrier and
environmental sources - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 98
Table 42 Antimicrobial resistance profile of 297 S aureus strains obtained from clinical,
carriage and environmental sources - - - - - - - 99
Table 43 Resistance phenotype of S aureus isolates from clinical, carriage and environmental
sources - - - - - - - - - - - - - 100
Table 44 Antimicrobial resistance profile of MRSA - - - - 101
Table 45 Antimicrobial susceptibility profile of staphylococcal isolates - - - - - - - - 102
Table 46 Distribution of virulence determinants in S aureus isolates from carrier, clinical and
environmental sources - - - - - - 114
Table 47 Comparison of virulence determinants in S aureus isolates from carrier, clinical and
environmental sources - - - - - - - - - - - 115
Table 48 Distribution of PVL positive S aureus isolates from clinical infections - - - - 116
Table 49 Association between agr groups and clinical infections - - - - - - - - 117
Table 410 Distribution of 37 spa types among S aureus strains from clinical, carriage and
environmental sources - - - - - - - - - - - - - - - 130
Table 411 Distribution of spa types in 8 different medical centres in Lagos and Ogun
States - - - - - - - - - 132
Table 412 Characterization of 297 S aureus strains by spa type antibiotic susceptibility pattern,
agr type and detection of virulence genes - - - - - - - - - - - 133
Table 413 Clonal characterization of 8 methicillin resistant S aureus strains by detection of
virulence determinants, agr type, spa type and SCCmec type - - - 135
LIST OF FIGURES
Figure 21 Typical colonies of S aureus - - - - - - - - 7
Figure 22 Pathogenic factors of S aureus, with structural and secreted products both playing
roles as virulence factors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 13
Figure 23 Model for the emergence of Panton-Valentine Leukocidin (PVL)â€"producing
community-associated methicillin-resistant S aureus (MRSA) - - 31
Figure 24 Induction of staphylococcal β-lactamase synthesis in the presence of the β-lactam
antibiotic penicillin - - - - - - - - 35
Figure 25 Comparison of S aureus SCCmec types - - - - - - - - - - - - - 39
Figure 26 Schematic of PCR - - - - - - - - - - - - - - - 61
Figure 31 Sources of clinical isolates - - - - - - - - - - 72
Figure 41 Dendrogram showing relationship between 8 MRSA strains based on analysis of
antibiotic resistance - - - - - - - - 104
Figure 42 Comparison of antimicrobial resistance patterns of MRSA and MRCoNS - - - - 105
Figure 43 Dendrogram showing relatedness of S aureus to coagulase negative staphylococci
based on antibiogram - - - - - - - - - 106
Figure 44 Distribution of virulence factors in 297 S aureus isolates - - - - - - 118
Figure 45 Quantification
