Molecular Characterization and Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Clinical and Environmental Sources

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Molecular Characterization and Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Clinical and Environmental Sources

By

AYEPOLA, OLAYEMI OLUSEUN

Presented To

Department of Biological Science

256 Pages 60,138 Words 2,428 Views PDF Format No. of Chapters: 1 to 5 Chapters

ABSTRACT

Staphylococcus aureus is an important pathogen causing skin and soft-tissue infections, systemic infections and toxemic syndromes. In order to have adequate information for treatment of S. aureus infections, it is important to understand trends in the antibiotic-resistance patterns as well as clonal identities across geographical regions. A total of 297 non-duplicate S. aureus isolates (209 clinical, 84 carrier and 4 environmental) were characterized by phenotypic and genomic methods. Antimicrobial susceptibility testing was performed by disk diffusion and the automated VITEK-2 system. PCR was used to amplify genes for accessory gene regulator (agr); capsular polysaccharide (cap) 5 and 8, exfoliative toxins (eta and etb), the toxic shock syndrome toxin- 1(tst) and Panton-Valentine Leukocidin (PVL). Typing of isolates was by the staphylococcal protein A (spa) typing. High level resistance was observed against penicillin and ampicillin (97.3%); trimethoprim/sulfamethoxazole (80%) and tetracycline (17.5%). Azithromycin, clarithromycin, erythromycin, clindamycin, linezolid, vancomycin, nitrofurantoin, fusidic acid, mupirocin and rifampicin recorded 100% activity against the isolates. Ninety-five percent of all strains (n=281) harboured the Î²-lactamase (blaZ) gene and 2.7% (n=8) possessed the mecA gene. The methicillin resistant S. aureus (MRSA) strains were resistant to at least 10 antibiotics including all penicillins, penicillin/penicillinase inhibitor combinations, carbapenem and cephalosporins. The staphylococcal cassette chromosome mec (SCCmec) typing of MRSA strains detected only SCCmec types I and IV in two strains (Y260: type I and Y59: type IV). The eta and tst genes were present in 0.7% (n=2) and 1.7% (n=5) of S. aureus isolates respectively. A high prevalence of PVL genes was noted in clinical isolates (79.4%; n=166); carrier isolates (56%; n=47) and environmental isolates (75%; n=3). The PVL protein was expressed in vitro by 68.5% of strains harboring lukS-PV and lukF-PV gene. All strains carried either the cap8 (91.9%; XX n=273) or cap5 locus (7.7%; n=23) while one MRSA strain was untypeable. A Single agr allele was detected in each S. aureus isolate with the majority in agr-2 (73.4%; n=218). Thirty-seven spa types were identified; predominant spa types among the methicillin-susceptible S. aureus (MSSA) were t084 (65%), t2304 (4.4%) and t8435 (4%). Prevalent spa types in MRSA were t002, t008, t064, t194, t8439, t8440 and t8441. Eleven novel spa types (t8435, t8436, t8437, t8438, t8439, t8440, t8441, t8442, t8952, t8953, t8953) were identified. The pT181 plasmid was successfully used to confer tetracycline resistance in S. aureus strains A56 and Y1. The use of phenotypic and molecular methods in this study provided useful information on antibiotic resistance and genetic diversity of S. aureus isolates from Ogun and Lagos States of Nigeria. The information provided could help in monitoring the evolution of S. aureus strains in Nigeria over time.

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   Î²-lactam antibiotics - - - - - - - 33
272 Penicillins - - - - - - - 33
273 Penicillinase-stable Î²-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 Î²-lactamase synthesis in the presence of the Î²-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

256 Pages 60,138 Words 2,428 Views PDF Format No. of Chapters: 1 to 5 Chapters

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