ABSTRACT
Staphylococcus aureus (S. aureus) is a unique microorganism among Staphylococcus spp notoriously recognized globally for its clinical importance in causing clinical or subclinical bovine mastitis in livestock. In humans, it causes food poisoning, toxic shock syndrome, scalded skin disease and bacteraemia as an invasive complication, which may result to osteomyelitis, endocarditis, boils, cellulitis, pneumonia and thrombophlebitis among others. The dissemination of S. aureus and its variant methicillin resistant S. aureus (MRSA) between different animal species has been documented in many developed countries especially in regions of high dairy farming, pointing out livestock associated MRSA (LA- MRSA), community affiliated MRSA (CA-MRSA) and hospital affiliated MRSA (HA- MRSA) which may freely be transmissible between domesticated and wild animals, poultry and humans. The aims of this study included: isolation and identification of S. aureus from blood of human patients and raw cow milk, determination of antimicrobial susceptibility (AST) patterns of S. aureus from human blood and raw dairy milk from selected farms in peri-urban Nairobi, to determine and compare resistant phenotypes of S. aureus strains isolated from milk against those isolated from blood of human patients and to determine the various genetic determinants for MRSA strains and thereafter undertake sequencing g of resistant genotypes. The study used convenience sampling strategy, in a one off sampling process employing inclusion and exclusion criteria. The samples were collected between November 2016 and October2017. A total of 353 milk samples and 142 human blood samples were collected employing aseptic techniques and transported to the University of Nairobi, Department of Public Health, Pharmacology and Toxicology for S. aureus isolation and characterization. Isolation and identification of coagulase positive (COPs) S. aureus was done by selective media, namely Mannitol salt agar (MSA) and coagulase testing using reconstituted rabbit plasma and then genotypically confirmed by Polymerase chain reaction (PCR) using specific primers for nuc (thermonuclease) gene of S. aureus. AST of S. aureus isolates was done by disk diffusion method employing Clinical and Laboratory Standards Institute (CLSI), 2017 guidelines using S. aureus ATCC 25923 as standard reference organism. A panel of 8 antibiotics were used for AST; cefoxitin 30g (as the surrogate antibiotic for methicillin), ampicillin 10g, gentamycin 10g, ciprofloxacin 5g, amoxicillin-clavulanic acid 30g, erythromycin 15g, tetracycline 30g and trimethoprim /sulfamethoxazole 1.25/23.75g. The diameters of zones of inhibition were measured to closest whole millimetre, then the interpretative criteria for each antimicrobial agent was determined using the criterion described by CLSI, 2017. The isolates that tested resistant phenotypically to cefoxitin 30g were genotypically identified through amplification of the nuc gene, mecA gene and mecC gene by PCR specific primer pairs. PCR assay was employed to detect resistant determinants (mecA and mecC) genes, which are linked to conferring methicillin resistance. The PCR amplicons were electrophoresed on 1.5 % agarose gel in Tris-acetate-EDTA buffer containing 0.5µg/ml of ethidium bromide using 100 bp DNA ladder. The gels were viewed and documented using UV transilluminator digital camera (Gelmax 125 imager, Cambridge UK) with UVP software interphase computer (Upland CA, USA) Positive samples identified through amplification of mecA gene and mecC had their PCR products, alongside their specific primers (both forward and reverse) previously used for identification of the resistant genes with their PCR products were submitted to Humanizing Genomics, Macrogen Europe Laboratory- Netherlands for sequencing. The Basic local alignment search tool (BLAST) of the NCBI Gene bank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to align the PCR product sequences for similarity check after being read by the Gene Runner software. The BLAST results were employed in confirmation of S. aureus isolates harboring the assayed resistant genes, by alignment with the homologue in the gene bank, which were then linked to the host from which the resistant isolate was obtained. S. aureus prevalence in raw milk and human blood was 7.4 % and 37.3 % respectively. The S. aureus isolates from raw milk were resistant to the following panel of antibiotics; cefoxitin, (11.54%), ampicillin, (15.38%), erythromycin, (3.85%), gentamycin, (7.69%), tetracycline, (15.38%) and sulfamethoxazole-trimethoprim (3.84%) and no resistance were noted to amoxicillin/clavulanic acid and ciprofloxacin. Isolates of S. aureus from human blood, had cefoxitin resistance marked at 20 (37.74%), ampicillin 27 (50.94%), ciprofloxacin 15 (28.3%), erythromycin 10 (18.87%), gentamycin 15 (28.30%) tetracycline 19 (35.85), amoxicillin-clavulanic acid 15(28.30%) and sulfamethoxazole-trimethoprim 29 (54.72%) while erythromycin, gentamycin, and sulfamethoxazole-trimethoprim showed intermediate resistance of 9 (16.98%), 1(1.89%) and 2 (3.77%) respectively. Overall MRSA prevalence among confirmed S.aureus isolates from cow milk and human blood was 11.54 % and 37.74 % respectively. All the three S. aureus isolates from milk that were phenotypically methicillin resistance did not expressed mecA or mecC genes by PCR assay while S. aureus isolates (n=20) from human blood, 17 (85 %) expressed mecA gene and 3 (15 %) isolates did not express mecA nor mecC genes and 22.64 % expressed gyrA gene, 24.53 % expressed gyrB gene and 18.87 % expressed tetM gene by PCR. On the other hand, 4 (15.38 %) of the 26 isolates from raw cow milk expressed tetM gene and the three isolates of S. aureus that were phenotypically methicillin resistant did not express mecA nor mecC genes by conventional PCR assay. This study noted an overall difference in resistance of S. aureus strains from humans to nearly all-antimicrobial classes as compared to S. aureus isolates from raw milk. Multidrug resistance was observed among 19 (35.84%) isolates of S. aureus from human blood. The most frequent MDR phenotypes for S. aureus identified in this study were; FOX- AMP-CIP-GENT-AMC-SXT and FOX-AMP-CIP-ERY-GENT-TET-AMC-SXT in 8(15.09%) and 4 (7.55%) of isolates respectively (Table 4.6). In total, 9 (16.98%) MDR phenotypes were identified; FOX-AMP-CIP-ERY-GENT-TET-AMC-SXT (7.55%), FOX- AMP-CIP-ERY-GENT-AMC-SXT (1.88%), FOX-AMP-CIP-GENT-AMC-SXT (15.09%), FOX-AMP-CIP-ERY-GENT-AMC (1.88%), FOX-AMP-CIP-GENT-AMC (1.88%), FOX-AMP-SXT (1.88%), AMP-TET-SXT (1.88%), AMP-ERY-SXT (1.88%) and ERY-TET-SXT (1.88%). Different resistant phenotypes and their corresponding genetic determinants for resistance of the MDR-S. aureus isolates from human blood were detected in this study. About (78.95 %) of the multidrug resistant isolates from human blood were MRSA and 73.68 % of MDR-S. aureus harbored mecA, 63.16 % gyrA, 68.42 % gyrB and 26.32 % tetM. Our results indicate that mecA gene was the predominant genetic determinant for methicillin resistance phenotypes, followed by gyrB, gyrA and tetM for the resistance of ciprofloxacin and tetracycline respectively. There was no multidrug resistance (MDR) noted among the isolates of S. aureus from raw milk but seven resistant phenotypes were evident; TET (7.69%), FOX-GENT (3.85%), AMP-TET (3.84%), ERY- SXT (3.84%), AMP (11.54%), FOX (7.69%), and GENT-TET (3.85%) wherein; AMP and FOX and TET were the top three frequently identified phenotypes. In this study, S. aureus was identified to be present in both dairy milk and human blood. Ciprofloxacin, amoxicillin / clavulanic acid and trimethoprim/sulfamethoxazole were the most effective agents against S. aureus isolates from cattle while ciprofloxacin, erythromycin and amoxicillin / clavulanic acid were the most effective against S. aureus isolates from humans. The study further shows that low to moderate MRSA phenotypes were observed in both cattle and humans, however MRSA strains from human isolates were three folds more than that from cattle. Also the study shows that MRSA isolates from humans harboured mecA gene while the isolates from cattle did not express mecA nor mecC genes. The resistant determinant mecA gene in human blood in the current study was alike to some strains of MRSA from animals in other parts of the world and therefore demonstrating the zoonotic pontential of this resistant gene.
TABLE OF CONTENTS
DECLARATION x
DEDICATION xi
ACKNOWLEDGEMENTS xii
LIST OF TABLES xvii
LIST OF FIGURES xviii
LIST OF APPENDIX xix
ABBREVIATIONS AND ACRONYMS xx
ABSTRACT xxii
CHAPTER ONE: INTRODUCTION
1.1 Background information 1
1.2 Statement of the problem 6
1.3. Justification 7
1.4 Hypothesis 8
CHAPTER TWO: LITERATURE REVIEW
2.0 Description and Taxonomy of Staphylococcus aureus 9
2.1 Distribution of Staphylococcus aureus 12
2.2. Diseases caused by Staphylococcus aureus in animals 13
2.3. Diseases caused by Staphylococcus aureus in humans 14
2.4. Methicillin Resistant Staphylococcus aureus (MRSA) 16
2.4.1 Types of MRSA 19
2.4.1.1 Hospital acquired MRSA (HA-MRSA) 19
2.4.1.2 Community acquired MRSA (CA-MRSA) 20
2.4.1.3 Livestock associated MRSA (LA-MRSA) 21
2.5 MRSA between animals and humans 22
2.6 Global distribution of MRSA 24
2.6.1 Epidemiology of MRSA in Europe 25
2.6.2 Epidemiology of MRSA in United States of America 27
2.6.3 Epidemiology of MRSA in Asia 28
2.6.4 Epidemiology of MRSA in Africa 29
2.6.5 Epidemiology of MRSA in Kenya 31
2.7 Virulence mechanisms for MRSA 31
2.8 Laboratory methods of diagnosing MRSA strains 32
2.9 Classification of βeta lactam antibiotics 35
2.10 Mechanisms of action of βeta-lactam antibiotics 37
2.11 Mechanisms of antibiotic resistance 37
2.11.1 Resistance mechanisms to βeta-lactam antibiotics 38
2.11.2 Tetracycline resistance 40
2.11.3 Chloramphenicol resistance 41
2.11.4 Aminoglycoside antibiotics resistance 41
2.11.5. Macrolides, Lincosamides and Streptogramin (MLS) resistance 43
2.11.6 Quinolone Resistance 44
2.11.7 Sulfonamides and Trimethoprim Resistance 45
2.11.8 Multidrug Resistance (MDR) S. aureus 46
CHAPTER THREE: MATERIALS AND METHODS
3.1 Reference Bacteria 48
3.2 Chemicals, Glassware, Media and Plastic ware 48
3.3 Equipments 48
3.4 Study Sites 49
3.4.1 Human blood sample 49
3.4.2 Dairy milk samples 50
3.5 Study Design 51
3.6 Sample Collection and Handling 53
3.6.1 Milk sample collection 53
3.6.2 Human blood samples 53
3.6.3 Handling of the samples Error! Bookmark not defined.
3.7 Bacterial isolation and identification 54
3.7.1. Isolation of bacteria from milk samples 54
3.7.2. Isolation of bacteria from blood samples 54
3.8 Biochemical Test for S.aureus 55
3.8.1 Slide Catalase test 55
3.8.2 Tube coagulase test 55
3.9 Hemolytic activity 55
3.10 Preservation of S. aureus isolates 56
3.11 Antimicrobial susceptibility testing 56
3.11.1 Interpretation for Antibiotic sensitivity 57
3.11.2 Detection of MRSA by disk diffusion 58
3.12 Molecular identification of S. aureus and PCR Detection of mecA 58
3.12.1. Extraction of DNA 59
3.12.2 Confirmation of S. aureus isolates 59
3.12.3 Detection of mecA gene by PCR technique 60
3.12.4 Detection of mecC gene by PCR technique 61
3.13 Gel Electrophoresis of PCR products 62
3.14 Sequencing of Resistant genes 63
3.15 Bioinformatic analysis 63
3.16 Data analysis 63
CHAPTER FOUR: RESULTS
4.0. S. aureus isolation and identification 65
4.1 Culture and Biochemical for dairy Milk samples 65
4.2. Confirmation of S. aureus isolates by PCR 67
4.3 Antimicrobial susceptibility testing 69
4.3.1 Susceptibility patterns of S.aureus isolates from raw cow milk and human blood 70
4.3.2 Resistance of S.aureus isolates from raw cattle milk and human blood to β- lactam and to non β-lactam antibiotics 71
4.3.3 Resistance phenotypes of S. aureus isolates from raw cow milk and human blood to eight (8) different classes of antimicrobial agents 73
4.4. Genetic determinants responsible for antimicrobial resistance phenotypes 74
4.5 BLAST analysis of DNA sequences 77
4.5.1 Identification of DNA sequences for resistant gene 77
CHAPTER FIVE: SUMMARY CONCLUSION AND RECOMMENDATIONS
5.2. Conclusion from this study 97
5.3. Recommendations from this study 99
REFFERENCES 100
APPENDICES 139
LIST OF TABLES
Table 2.1 Summary of the classification of Staphylococcus aureus 11
Table 2.1: Classification of β-lactam antibiotics, examples and spectrum of activity 36
Table 3.1: Interpretive category and Zone Diameter Breakpoints (CLSI, 2017) 57
Table 3.2: Primers used in the detection of thermo-nuclease (nuc) gene, mecA, mecC,gyrA, gyrB and tetM genes 59
Table 4.1: S. aureus isolates recovered on culture and confirmed as positive isolates of S. aureus using biochemical test 67
Table 4.2: Nuclease genes of S. aureus and their sequenced homologue and identity obtained from NCBI genebank using Nucleotide-nucleotide BLASTn 69
Table 4.3: Resistance of S.aureus isolates from raw cattle milk and human blood to eight different antimicrobial agents 71
Table 4.4 Resistance pattern of S.aureus isolates from raw cow milk and human blood to some β-lactams and to non β-lactam antibiotics 72
Table 4.5. Proportion of antimicrobial resistant phenotypes of S. aureus isolates from human blood, including multidrug resistant S. aureus 74
Table 4.6 Multidrug-Resistant genes of S. aureus 76
LIST OF FIGURES
Fig. 2.1 Golden- yellow colonies of S. aureus on Mannitol Salt Agar 10
Fig. 2.2 S. aureus on a blood agar plate 11
Fig. 2.3 Quotient of S. aureus isolates from blood resistant to methicillin in countries participating in EARSS, 2002 (EARSS Annual Reports). 26
Fig. 3.1: Map showing Mukuru Kwa Njenga slums 50
Fig. 3.2: Map showing Peri Urban Nairobi 51
Fig. 3A- 4D: Isolation and identification of S. aureus suspects 66
LIST OF APPENDICES
Appendix 1: Clinical and Laboratory Standards Institute (CLSI) Guideline M100, Table 2C. Zone Diameter and Minimal Inhibitory Concentration (MIC) Breakpoints for Staphylococcus spp. 139
Appendix 2: Regional distribution of MRSA resistance to β-lactam antibiotics globally. 141
ABBREVIATIONS AND ACRONYMS
BA : Blood Agar
blaZ : β-Lactamase gene
Bp : Base pair
MRSA : Methicillin Resistant Staphylococcus aureus
CA-MRSA : Community acquired Methicillin Resistant Staphylococcus aureus
CC : Clonal complex
Ccr : Cassette chromosome
DNA : Deoxy ribonucleic acid
dNTP : Deoxy ribo nucleoside triphosphate
Fig. : Figure
G : Gram
h : Hour
HA-MRSA : Hospital acquired Methicillin Resistant Staphylococcus aureus
I.U : International Units
LA-MRSA : Livestock associated Methicillin Resistant Staphylococcus aureus
mecA : Mec A gene
mecC : Mec C gene
mg : milli gram
Mgcl2 : Megnesium chloride
MHA : Muller-Hinton agar
min : Minutes
Ml : Millilitre
MLST : Multilocus sequence type
Mm : Millimolar
MSA : Mannitol Salt Agar
Ng : Nano gram
Nm : Nano molar
PBP : Penicillin Binding Protain
PCR : Polymerase Chain Reaction
Rpm : Revolution per minute
SCC : Staphylococcal cassette chromosome
Spa gene : Staphylococcal protein A
Taq :Thermus aquaticus
TBE : Tris -Borate with EDTA
TSA :Tryptic Soya Agar
UV : Ultra Violet
µg : micro gram
µl : micro litre
CHAPTER ONE: INTRODUCTION
1.1 Background information
Staphylococcus aureus (S.aureus) is a primal agent belonging to Staphylococcaceae family group of organisms (CABI, 2020). The bacterium co-exists as a normal flora on different parts of the body including; pharynx, skin, intestine and vagina (Lowy, 1998). The micro-organism (S. aureus) uses complex regulatory mechanisms to sense varied stimuli to favorable conditions for growth and multiplication, pathogenicity and modulation of its virulence (Balasubramanian et al., 2017). It also possesses multiple toxins and virulent mechanisms (Lowy, 1998; Boswihi and Udo, 2018). The pathogenicity of S. aureus is enhanced through secretion of toxins like Panton-Valentine Leucocidin (PVL), 33-kd protein-alpha toxins, and exfoliatin A and B toxins (Lowy, 1998). These toxins pose a health threat to both humans and animals by causing various diseases of the skin including boils, folliculitis, carbuncles, impetigo and other related health tortuousness including toxic shock syndrome (TSS), mastitis and meningitis (Makgotho, 2009).
The bacterium is notoriously known to cause hospital and community invasive and soft tissue infections (Omuse et al., 2014; Boswihi and Udo, 2018). The organism is known to be sporadic in a wide compass of ecological habitats and specific parts of domestic animals like dog nose and also from blood and body surfaces of humans (Mbogori et al., 2013). In humans, the organism is responsible for a spectrum of diseases including osteomyelitis, bacteraemia, endocarditis, boils, skin abscesses, cellulitis and surgical site infections and also causes mastitis and septicaemia in dairy cattle, and arthritis in poultry (Mbogori et al, 2013).
Other species of Staphylococcus such as S. epidermidis is linked to causing infections related with indwelling medical devices, S. saprophyticus causes infections of the urinary tract system amongst young girls of adolescent age, whereas S. warneri, S. lugdunensis, S. schleiferi, S. intermedius and S. haemolyticus are inconsistently associated with health care setting pathogenicity (Makgotho, 2009).
Among the Staphylococcus spp., coagulase-positive (COPs) S. aureus and coagulase-negative (CNS) such as S. epidermidis and S. haemolyticus are of human and veterinary medical significance (Misic, et al., 2015; Bierowiec et al., 2019)
S. aureus is an important clinical pathogen due to its extracellular virulency that enhances its colonisation and pathogenicity after surpassing the host defence mechanism (Bien et al., 2011; Balasubramanian et al., 2017). Therapeutic management of diseases caused by S. aureus has become complicated to health care providers in attaining the intended outcomes due to the pathogen’s ability to develop multi-drug resistance (Lowy, 1998; Gnanamani, 2017; Gheorghe et al., 2019).
Prior to 1942, management of diseases caused by S. aureus involved the use of -lactam antibiotics such as penicillins (Makgotho, 2009). After the development of methicillin (semi synthetic penicillins) in the late 1950s and its introduction into clinical practice in 1959, (Chamber and Deleo, 2009; Lakhundi and Zhang, 2018) to manage diseases caused by S.aureus as an alternative therapy to natural penicillins (Lowy, 2003), strain of methicillin resistant S. aureus (MRSA) was detected in 1961, two years later, following unveilling of methicillin into market (Chambers, 2001) and in the late 1960s, 80 % of community and hospital affilliated S. aureus were reported to be resistant to to penicillin ( Lowy, 2003 ) after which numerous virulent multidrug resistant strains of S. aureus were evident,in UK (Jevons, 1961; Livermore, 2000; Lowy, 2003). After UK, MRSA isolates were reported from other parts of European countries as well as Africa viz. USA, Malaysia, Australia, North Africa and East Africa as well (Lakhundi and Zhang, 2018; Guo, et al., 2020).
In a decade after MRSA was reported (1961 to 1970), infections related to the pathogenic strain were confined to hospital (Lakhundi and Zhang, 2018) and currently predominantly established in the community (Lowy, 2003). In the early 1980s, the bacterium was noted as the leading causative agent for nosocomial infections (Makgotho, 2009; Lakhundi and Zhang, 2018) and a pattern of inter-transmission between hospital and community MRSA was noted (Makgotho, 2009).
Between 1993 and 2003, new MRSA strains phenotypically and genotypically distinctive from the native hospital acquired MRSA (HA-MRSA) was noted in community indicating phylogenesis of the native MRSA (Makgotho, 2009) and between 1960s and 2005, about 19,000 deaths directly linked to MRSA and 100,000 seriously ill of MRSA infections were reported worldwide (Egege et al., 2020). From the year 1987, community associated MRSA (CA-MRSA) increased tremendously causing clinical manifestions such as necrotising severe skin and soft tissue infections, pneumonia, and mastitis (Makgotho, 2009) until 1990s when HA-MRSA became pandemic (Johnson et al., 2005) with a doubled hospitalization as aresult of MRSA related infection between 1999 and 2005 (Egege et al., 2020). MRSA is a genetically distinctive strain S. aureus that expresses mecA gene or mecC gene and therefore conferring other resistance mechanisms such as change of affinity to penicillin-binding proteins for β-lactam antibiotics such the penicillins, carbapenems, monobactams and cephalosporins (Gunawardena et al., 2012; CLSI, 2017).
The morbidity and/or mortality related to S. aureus infections are very high and in this case, hospital acquired infections are more often related to antibiotic resistant pathogen (Okon et al., 2011). Even though wide range of antibiotics are available in medicine, researchers are still actively searching for a new antimicrobial agent with superior activity due to ability of bacteria becoming resistant to currently available antibiotics (Basak et al., 2015), for example 60 % of S. aureus strains are methicillin resistant, and some strains have also started becoming resistant to vancomycin (Patrick, 2013; Basak et al., 2015). Currently, the rising incidence of S. aureus resistance to vancomycin has spurred fear among the health care providers and this has become growing worldwide concern (Tiemersma et al., 2004).
MRSA contributes to a greater percentage of hospital and community acquired infections globally. It is estimated that in European Union over 15,000 people suffer from hospital acquired MRSA related infections and this has increased the burden of In-hospital costs of up to Euro 380 million and longer days of hospitalization (Köck et al, 2010). The emergence of multi-drug resistant bacterial strains in many healthcare systems that has narrowed the spectrum of effective antibiotic for clinically challenging infections has become a de-facto monopoly globally for premature deaths and extra days of hospitalization (Peters et al., 2019). Overall MRSA accounts for 44 % of heath-associated infections, 22 % of which are attributed to extra deaths and 40 % of extra days of hospitalization due to resistant pathogenic MRSA strain (Kot et al., 2020). Community acquired methicillin resistant S. aureus (CA-MRSA) strains has emerged to be a principal health concern globally since the late 1980’s when it was first reported in western Australia among communities with no previous records of hospitalization and currently documented evidence rests on colonized animals being reservoirs and shedders and possible transmission between human and animal species may not be ruled out (Boswihi and Udo, 2018). The carrier rate of S.aureus amongst healthy individual lies between 15 % and 35 % with 38 % risk of that individual developing an associated infection with an additional infection risk of 3 % when colonized with MSSA (File, 2008).
According to Dora (2011) MRSA prevalence rate in Europe stands at 26 % while in USA, 61.8% of the patients were colonized by MRSA strains and 38.2 % infected by MRSA related diseases (Jarvis et al., 2012). In Asia, S. aureus prevalence was noted to be above 60 % of which MRSA contributed for 25.5 % of all the community associated illnesses and 67.4% of hospital related sicknesses (Song et al., 2011). In Africa, data on MRSA is sparingly documented, however S. aureus prevalence rates vary from 5 % to 45 % (Dora, 2011) while Omuse et al., (2014) reported MRSA prevalence rates as low as 4 % and as high as 82 %. With the exception of Southwest parts of Africa, MRSA has been reported from most parts of African continent starting with Madagascar with MRSA prevalence rate of 5 %, Algeria 45 %, Tunisia 8.1 % respectively (Dora, 2011).
Among the East African countries, the MRSA prevalence ranges between 16 % to 27 % in which Tanzania reports a prevalence rate of 16 % and Kenya at a prevalence rate of 6.9 % (Aiken et al., 2014), However another survey conducted between 1996 and 1997 in eight African countries reported a varied reports of MRSA prevalence rates ranging from 20 % to 30% in Cameroon, Nigeria and Kenya but the prevalence rate in Tunisia and Algeria was less than 10% (Kesah et al., 2003). Bloodstream isolates of S. aureus at University Hospital in Kenya retrospectively reported MRSA prevalence rate of 21 % (Omuse et al., 2014).
In livestock, MRSA has become a disturbing concern because of its zoonotic nature, contamination of milk or dairy produts and it is associated high disease treatment costs (Keyvan et al., 2020) or rather serve as potential reservoirs for zoonotic infections (Fitzgerald, 2012). Various publications from different parts of the world have reported varied prevalence of MRSA in milk as in Iran 16.2 %, (Jamali et al., 2015), Italy 2.5 %, (Parisi et al., 2016), Italy 0.7 %, (Giacinti et al., 2017), Czech Republic 6.1 %, (Klimešová et al., 2017), Uganda 56.1 %, (Asiimwe et al., 2017) and Turkey 1.7 % (Ektik et al., 2017). In South Africa, 6% was reported in dairy milk in two commercial farms (Ateba et al., 2010) and in Nigeria, Omoshaba et al., (2020) reported MRSA prevalence of 18.5 % in raw milk, 37.7 % in sheep, 23.4 % in goats and 7.5 % from nasal swabs of small ruminants. Animals can transmit MRSA resistant strain not only holizontaly or vertically but also in raw dairy products intended for commercial processing and for consumption (Klimešová et al., 2017; Omoshaba et al., 2020).
1.2 Statement of the problem
While there is a wide range of antimicrobial agents available in medicine with improved antibacterial spectrum, there remains a worrying clinical concern over the continued possibility of bacteria to acquire resistance to these agents. Even though articles published on MRSA in Africa have reported various possible origins of MRSA strains (animals, animal products and humans); but these MRSA isolates from different independent setting of study have rarely been compared.
The incidences of epidemic strains of MRSA are increasing in many African countries and this situation is posing a feasible threat to available therapeutic agents and alternative options.
In Africa, including Kenya, the prevalence of MRSA clones is not well documented and therefore determining the antimicrobial resistance patterns may address this gap and challenge (Falagas et al., 2013).
1.3. Justification
Elucidating the prevalence of MRSA isolates among animal and human and assessing the phenotypic and genotypic characteristics of MRSA from human clinical isolates and raw cow milk from different regional settings, remains an important step towards curbing inter and cross transmission and upward trends in MRSA related infections in both humans and animals.
Healthcare policy makers on appropriate therapeutic interventions to clinically challenging conditions can use data on MRSA prevalence, generated from this study and facilitate national planning on attainable treatment protocols on common staphylococcal related infections so that an appropriate antimicrobial therapy is initiated for better therapeutic outcomes, with minimal attributable in-hospital costs.
Data on MRSA prevalence in raw milk from this study will help relevant professional body and authorities responsible for surveillance and prevention of bacterial resistance to antimicrobial agents to formulate policies and guidelines emphasizing on areas vulnerable to indiscriminate use of antibiotibics, considerable overlap between antibiotic agents consumed in human or veterinary medicine and overall consumption of antibiotics in animal production and implement antibiotic stewardship to protect animals and humans from the rising threat of antibiotic resistance.
1.4 Hypothesis
MRSA is not common in human blood and raw cow milk.
1.5. Objectives
a). General objective
To identify and determine molecular characterization of Methicillin Resistance S. aureus obtained from blood of human patients from Nairobi healthcare facilities at Mukuru slum and raw cow milk from selected farms in Peri-urban Nairobi.
b). Specific objectives
I. To isolate and identify the prevalence of S. aureus from blood of human patients and raw cow milk.
II. To determine the antimicrobial susceptibility profiles of S. aureus isolated from blood of human patients at community healthcare facilities and raw cow milk from selected dairy farms in peri-urban Nairobi, Kenya.
III. To determine and compare antimicrobial resistant phenotypes of S.aureus isolated from blood of human patients at community healthcare facilities with that isolated from raw milk from selected farms in peri-urban Nairobi, Kenya.
IV. To determine the genetic determinants responsible for MRSA strains.
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