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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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