TABLE OF CONTENTS
Title Page
Abstract
Table of Contents
List of Abbreviations and Symbols
CHAPTER ONE: INTRODUCTION
1.1 Background of the Study
1.2 Statement of Research Problem
1.3 Justification
1.4 Aim
1.5 Objectives
1.6 Research Questions
CHAPTER TWO: LITERATURE REVIEW
2.1 General Characteristics of Staphylococcus
2.1.1 Genome characteristics
2.1.2 Natural habitats and reservoirs of Staphylococcus species
2.1.3 Pathogenic members of the genus Staphylococcus
2.1.4 General characteristics and history of Staphylococcus aureus
2.1.5 Epidemiology and transmission of Staphyococcus aureus
2.1.6 Virulence factors of Staphylococcus aureus
2.1.7 Molecular diagnosis of Staphylococcus aureus
2.1.8 Treatment and antibiotic resistance
2.1.9 Mechanism of antibiotic resistance
2.2 Methicillin-Resistant Staphylococcus aureus (MRSA) Organisms
2.2.1 Community- associated MRSA in humans
2.2.2 Hospital – associated MRSA in humans
2.2.3 Resistance and virulence factors associated with MRSA
2.3 Transmission of MRSA
2.3.1 Transmission of MRSA in humans
2.3.2 Zoonotic nature of MRSA and transmission in animals
2.3.3 Transmission of MRSA through milk
2.4 Prevalence of MRSA
2.4.1 Prevalence of MRSA in humans
2.4.2 Prevalence of MRSA in Nigeria
2.4.3 Prevalence of MRSA in animals
2.4.4 Prevalence of MRSA in milk
2.5 Clinical Signs of MRSA Infections
2.5.1 Clinical signs of MRSA in humans
2.5.2 Clinical signs of MRSA in animals
2.6 MRSA Infections in HIV/AIDS Patients
2.7 Diagnosis of MRSA
2.7.1 Molecular diagnosis of MRSA
2.8 Treatment of MRSA Infections
2.8.1 Treatment of MRSA infections in humans
2.8.2 Treatment of MRSA infections in animals
2.8.3 Phage therapy against MRSA infections
2.9 Control and Prevention of MRSA Infections
2.9.1 Control and prevention of MRSA in humans
2.9.2 Control and prevention of MRSA infections in animals
2.9.3 Vaccines against MRSA infections
2.10 Preparation of Yoghurt
2.11 Preparation of Nono
CHAPTER THREE: MATERIALS AND METHODS
3.1 Study Area
3.2 Sample Size Determination
3.3 Sample Size
3.4 Sample Collection and Transportation
3.5 Materials Used
3.5.1 Media
3.5.2 Sugars
3.5.3 Antimicrobial disks
3.5.4 Tests kits
3.5.5 Materials for molecular detection
3.5.6 Other materials used
3.6 Isolation and Characterization of S.aureus from Fermented Milk “NONO” And Yoghurt
3.6.1 Isolation procedure for S. aureus
3.6.2 Identification of Staphylococcus
3.6.3 Conventional biochemical tests for Staphylococcus
3.6.4 Identification of isolates based on conventional biochemical tests
3.6.5 Microbact test of Staphylococcus
3.7 Determination of the Susceptibility of S.aureus to Some Antimicrobial agents
3.7.1 Disks diffusion tests for S.aureus
3.7.2 ORSAB test
3.8 Molecular Detection of Isolates Using Polymerase Chain Reaction
3.8.1 Protocol of DNA Extraction of S. aureus Isolates
3.8.2 Protocol for PCR Amplification of mecA gene
3.8.3 Protocol for determination of penicillin – binding protein (Pbp2a) among MRSA isolates
3.8.4 Protocol for the determination of betalactamase among MRSA isolates
3.9 Statistical Analysis
CHAPTER FOUR: RESULTS
4.1 Occurrence of Staphylococcus Species
4.2 Susceptibility of Isolates to Antimicrobial Agents
4.3 Detection of mecA Gene by Polymerase Chain Reaction
4.4 Detection of Pbp2a and Betalactamase
CHAPTER FIVE: DISCUSSION
CHAPTER SIX: CONCLUSION AND RECOMMENDATION
6.1 Conclusion
6.2 Recommendations
REFERENCES
APPENDICES
ABSTRACT
Contamination of food products such as milk by Staphylococci, have been known to occur post pasteurization. Poor sanitary practices have also contributed to gross contamination. Indiscriminate use of antibiotics in efforts to treat diseases has led to the emergence of resistant strains known as MRSA. This study was to determine the presence of Staphylococcus including the methicillin resistant strains in yoghurt and nono. Bacteriological and molecular techniques were carried out to determine the occurrence of Staphylococcus aureus. Resistance was determined by detection of the mecA gene by PCR, betalactamase and altered penicillin -binding protein (Pbp2a).Out of 560 samples of yoghurt (280) and nono(280) collected from Kaduna and Zaria, the overall prevalence of Staphylococcal species were 75(13.39%) . Out of this 52(69.3%) were presumptively identified as methicillin resistant using the Oxacillin resistant staphylococcal basal medium (ORSAB) while 73(97.3%) were methicillin resistant by the Kirby-Baeur disk diffusion method. The 75 staphylococcal isolates were further subjected to Microbact 12S identification system and 17(3.04%) were confirmed as
Staphylococcus aureus.Out of this 12(70.59%) were presumptively identified as MRSA using ORSAB while 17(100%) were identified as MRSA using the Kirby-Baeur disk diffusion. The overall prevalence of MRSA was (3.04%). The occurrence of
Staphylococcus was higher in Zaria 41(14.64%) than in Kaduna (12.14%) while prevalence of MRSA was (3.21%) and (2.86%) in Kaduna and Zaria respectively. There was no statistical significant difference between the occurrence of these organisms and the locations (P˃0.05).The presence of the organisms was higher in nono 43(15.36%), and 12(4.29%) than in yoghurt 32(11.43%) and 5(1.79%) for Staphyloccocci and MRSA respectively but was not statistically significant (P˃0.05).All the isolates were resistant to more than one antibiotic but none resistant to all. The isolates of staphylococci and MRSA showed over 90% resistance to beta lactams such as penicillin, methicillin and oxacillin. The least resistance was to amikacin 34 (45.3%) and cefixime 7(41.2%) by staphylococcal species and MRSA respectively indicating that amikacin and cefixime are the drugs of choice. Resistance by the isolates was by a mec A independent mechanism. Eleven (64.7%) out of the 17 MRSA were positive for the penicillin – binding protein out of which 4(36.4%) were from yoghurt and 7(63.64%) from nono while 13(76.5%) were positive for betalactamase production out of which 4(30.77%) were from yoghurt and 9 (69.23%) from nono. The findings of this study, suggest that yoghurt and nono maybe vehicles for transmission of MRSA. Food safety should be advocated by relevant agencies and hygiene should be encouraged and enforced among the producers and sellers.
CHAPTER ONE
INTRODUCTION
1.1 Background of the Study
Milk accounts for 16% of the total volume of all food products produced from livestock in sub-Saharan Africa (F.A.O., 1986). Fresh milk and its fermented products (nono, yoghurt, kindirmo) constitute good media for microbial multiplication (Jawatz et al., 1991) and hence for transmission of milk borne diseases such as staphylococcosis, salmonellosis, brucellosis and tuberculosis among others.
Studies approximate that 30-50% of the human population harbour Staphylococcus aureus on their bodies (Sowash and Uhlemann, 2014). It can survive for hours on dry environmental surfaces (Whitt and Salyers, 2002).
S. aureus produces a variety of extracellular protein toxins and virulence factors which include pyrogenic exotoxins such as staphylococcal enterotoxins (SE), extracellular toxins, toxic shock syndrome toxin1 (TSST-1), exfoliative toxins that are implicated in the staphylococcal scalded skin syndrome (SSSS) in infants, alpha toxins, beta toxins, delta toxins and bicomponent toxins like Panton valentine leukocidin which is associated with severe necrotizing pneumonia in children. The SE are proteins which when ingested induce gastroenteritic syndrome in humans and can cause toxic shock syndrome (Marrack and Kappler, 1990). They are aetiological agents of soft tissue infections and also isolated from anterior nares of healthy individuals. S. aureus cause several diseases such as suppurative disease, mastitis, arthritis and urinary tract infection in animals such as dogs, cats and horses and can cause bumble foot in chickens. In........
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