ABSTRACT
Staphylococcus aureus, ‘one of the most adaptable and virulent pathogens in modern times’ is a facultative anaerobic Gram-positive coccal bacterium able to infect any tissue of the human body to cause ailments ranging from minor skin infections to life-threatening diseases. The bacterium is also known to colonize and infect both pets and livestock, including dogs, cats, rabbits, horses, cattle and pigs to result in zoonosis. A major concern is the prevalence of irrepressible multidrug resistant S. aureus in pets and livestock, as these may serve as reservoirs for human colonization and compound the problem of emergence and/or re-emergence of global diseases. This project was designed to assess the prevalence of multidrug resistant livestock associated S. aureus isolated from nasal passage of healthy cattle in Kara Market, Ogun state, Nigeria.
A total of 500 nasal samples were collected from healthy adult cattle (250 from male and 250 from female cattle) but 409 S. aureus isolates were identified(218 from male and 191 from female cattle) by Gram staining and specific biochemical tests.
The antibiogram of the S. aureus isolates revealed that all the isolates exhibited multidrugresistance (8/12) to ceftriaxone, gentamicin, cotrimoxazole, erythromycin, amoxicillin, streptomycin, chloramphenicol and ampicillin, fairly resistant(3/12) to quinolones but weakly resistant (1/12) to vancomycin. High minimum inhibitory concentration greater than 128 μg/ml was exhibited by 63.3% of the isolates against flucloxacillin (methicillin) but 28% of the isolates were equally resistant (>128 μg/ml) to vancomycin (glycosidic). The in-vitro combination effect of flucloxacillin and ampicillin on the isolates resulted in 86.3% additive to imply the unsuitability of the combination to effectively control staphylococcal infections.
Vancomycin was observed to be bactericidal to livestock associated multidrug and/or methicillin resistant S. aureus in this study. The quinolones antibiotics (ciprofloxacin, pefloxacin and ofloxacin) are fairly effective (known to be relatively non-toxic) may be used to complement and/or substitute toxic antibiotics in curtailing ailment resulting from multidrug resistant S. aureus.
Keywords: S. aureus, Antibiotics, Cattle, Resistance, Multidrug.
Word Count: 316
TABLE OF CONTENTS
Content Page
Title page i
Certification ii
Dedication iii
Acknowledgments iv
Abstract v
Table of Contents vi
List of Tables ix
List of Figures x
List of Plates xi
CHAPTER ONE: INTRODUCTION
1.1 Background to the Study 1
1.2 Statement of the Problem 3
1.3 Objective of the Study 3
1.4 Research Questions 4
1.5 Significance of the Study 4
1.6 Justification for the Study 5
CHAPTER TWO: REVIEW OF LITERATURE
2.1 The Genus Staphylococci 6
2.2 The Species Staphylococcus aureus 6
2.3 Cell Structure and Metabolism 8
2.4 Virulence Factors 9
2.5 Exotoxins 12
2.6 Superantigen Toxin 12
2.7 Mechanisms of Resistance 13
2.7.1 Penicillin Resistance 13
2.7.2 Methicillin Resistance 14
2.7.3 Quinolone Resistance 15
Content Page
2.7.4 Vancomycin Resistance 16
2.8 Livestock Associated Staphylococcus aureus and Livestock
Associated MRSA 18
2.9 The Problem of Resistance 23
CHAPTER THREE: METHODOLOGY
3.1 Research Design 26
3.2 Population 26
3.3 Sample size and sampling Technique 26
3.4 Sample Processing 27
3.5 Identification of the Isolates 28
3.5.1 Gram Staining 28
3.5.2 Microscopy 28
3.5.3 Catalase Test 28
3.5.4 Slide Coagulase Test 29
3.6 Antibiotic Sensitivity Test 29
3.6.1 Preparation of Antibiotic Solution for Disc Impregnation 30
3.6.2 Inoculum Turbidity Standard 30
3.7 Preparation of Antibiotic Solution for Minimum Inhibitory
Concentration 31
3.8 Determination of Minimum Inhibitory Concentration (MIC) 31
3.9 Synergism, Antagonism and Additive 32
3.8 Multiple Antibiotic Resistance Index and Statistical Analysis 32
CHAPTER FOUR: DATA ANALYSIS, RESULTS AND
DISCUSSION OF FINDINGS
4.1 Results 33
4.1.1 Identification and Distribution of S. aureus 33
4.1.2 Antibiotic Sensitivity Testing 36
4.1.3 Multiple Antibiotic Resistance Index 41
4.1.5 Minimum Inhibitory Concentration (M.I.C) 44
Content Page
4.1.6 Invitro Combination of Antibiotics 49
4.2 Discussion 51
4.2.1 Introduction 51
4.2.2 Distribution and attribute of Staphylococcus aureus 51
4.2.3 Antibiotic Sensitivity Testing 51
CHAPTER FIVE: SUMMARY, CONCLUSION
AND RECOMMENDATIONS
5.1 Summary 55
5.2 Conclusion 56
5.3 Recommendations 56
5.4 Contribution to Knowledge 56
REFERENCES 57
LIST OF TABLES
Table Page
4.1: Antimicrobial resistance and susceptibility data of S. aureus isolates 37
4.2:Multiple Antibiotic Resistance Index data of S. aureus isolated from
nasal passage of both male and female cattle. 42
4.3: Effect if invitro combination of antibiotics 50
LIST OF FIGURES
Figure Page
4.1: Source distribution and percentage of S. aureus in this study. 34
4.2: Drug resistance pattern of S. aureus isolated from nasal passage of healthy
female and male cattle. 38
4.3: Susceptibility pattern of S. aureus isolated from nasal passage of healthy
female and male cattle to fluoroquinolones. 39
4.4:MAR Index of S. aureus isolated from nasal passage of healthy male and
female cattle. 43
4.5:Minimum Inhibitory Concentration (%) of all S. aureus isolates to
flucloxacillin (μg/ml) by agar dilution. 45
4.6:Susceptibility pattern of S. aureus isolated from nasal passage of healthy
female and male cattle to varying concentrations of vancomycin by disc diffusion. 46
4.7:Minimum Inhibitory Concentration of all S. aureus isolates to
vancomycin (μg/ml) by agar dilution. 48
LIST OF PLATES
Plate Page
4.1: Fermentation of mannitol by S. aureus 34
4.2:Zone of inhibition of Fluoroquinolones amongst other antibiotics on
- aureus streaked Mueller-Hinton agar plate. 40
4.3: Zone of inhibition of varying concentrations of vancomycin. 47
CHAPTER ONE
INTRODUCTION
1.1 Background to the Study
Cattle are large bodied ruminants that feed on pastures and forages or fodder. In Nigeria, cattle are reared primarily for meat which is a veritable source of protein for humans, and for milk (Arowolo et al., 2013). Cattle rearing in Nigeria is an old occupation which is traditionally practiced by Fulanis and Shuwa Arabs in northern Nigeria with an extremely few local villages in the southern Nigeria (Erebor, 2003).
Staphylococcus aureus is a facultative anaerobic gram-positive coccal bacterium and due to a combination of numerous bacteria immune-evasive strategies which it uses, it is considered a successful pathogen. The nasal passages is considered to be the major habitat (Kluytmans et al., 1997; Lowy, 1998; Lowy, 2003) and the biggest supply of S. aureus in people, yet numerous body locales can harbor this bacterium (Vandenbergh & Verbrugh, 1999). S. aureus is a typical tenant of the skin (Lowy, 2003; Williams, 1963), perineum and can likewise be found in the axillae (Ridley, 1959), vagina (Guinan et al., 1982) and the gastrointestinal tract (Williams, 1963). S. aureus strains are noteworthy human pathogens and are conceivably ready in contaminating any human body tissue, bringing on everything from skin contaminations to life-debilitating sicknesses. In people, the diseases brought on by S. aureus can be partitioned into these three sorts in general; shallow sores, (for example, surgical site and wound contaminations), life and systemic undermining factors, (for example, osteomyelitis, endocarditis, pneumonia, mind abscesses/wounds, bacteraemia and meningitis), then toxinoses, (for example, poisonous stun disorder, sustenance harming and singed skin disorder (Alo et al., 2013; Aires de Sousa et al., 2004; Lowy, 2003). The sign of staphylococcal contamination are the boils that contain discharge which is made up of dead neutrophils, dead and living microbes, tissue (necrotic), the lysed host substance and bacterial cells. The immunocompetent hosts, as a rule, effectively clear the disease and deplete the ulcer, though for the immunocompromised and sporadically for a sound individual, the contamination might advance to a more profound tissues and turn into a conceivably lethal intrusive contamination (Norvick, 2006). It is still one of the five most common causes of nososcomial infections, often causing postsurgical wound infections (Bowersox, 1999). S. aureus is likewise known to colonize and contaminate both pets and animals, including pooches, felines, rabbits, stallions, steers and pigs (Morgan, 2008). A noteworthy concern is the nearness of methicillin safe S. aureus (MRSA) in pets and domesticated animals, as these may fill in as repositories for human colonization, an illustration is MRSA ST398 from pigs (Weese, 2010).
The unnecessary utilization of antibiotics has prompted to the rise of different medication safe strains of S.aureus (Lowy, 1998). The Penicillin was presented for curing infections caused by S. aureus in the 1940s, and adequately diminished mortality and bleakness. Be that as it may, in late 1940s, its resistance because of the nearness of penicillinase developed (Eickhoff, 1972). The staphylococci are extremely fit for advancing imperviousness to the regularly utilized antimicrobials, for example, erythromycin (Walmark & Finland, 1961), ampicillin (Klein and Finland, 1963), and antibiotic medication (Eickhoff, 1972). Much of the time, imperviousness to antimicrobial agents is coded for by qualities carried on plasmids, representing the quick spread of resistant microscopic organisms (Morris et al., 1998). One purpose behind the proceeding with essential part of S. aureus in illness is its inclination and propensity to wind up distinctly impervious to antimicrobial (Waldvogel, 2000). S. aureus is presently the main general reason for nosocomial diseases and, as more patients are dealt with outside the healing center setting, is an expanding worry in the group (CDC NNIS System, 2001; Diekema, 2001).
The time of medication development and its execution in human and creature wellbeing and horticulture was started by the revelation of anti-infection agents over 70 years prior. These disclosures were powerful against organisms consequently were viewed as effective against pathogenic microorganisms however this achievement was fleeting as they were tempered with in all cases by the rise of resistant microorganisms (D’Costa et al., 2011). A standout amongst the most relentless issues confronted by human services benefits far and wide is the expanding pervasiveness of antimicrobial resistance. This resistance is broadly perceived as a noteworthy general wellbeing danger and this issue is aggravated by a consistent reducing of the quantity of new specialists (antimicrobials) entering the clinical practice (D.H, 2000). There is an expanding worry that some less-alarming infections which were effortlessly treated are currently turning out to be progressively hard to treat and ailments created by microscopic organisms which are impervious to antimicrobial agents may set aside a more drawn out time of opportunity to treat successfully (Butler et al., 2006). In spite of the fact that the issue of multidrug resistance has pulled in the consideration of medicinal services administrations and the overall population, rates of antimicrobial resistance among healing center and group pathogens have expanded alarmingly amid the previous decade (NNIS, 2001).
1.2 Statement of the Problem
In Nigeria, cattle are reared primarily as a source of meat. According to Kuehnert et al. (2006), Lowy (1998, 2003), Onanuga & Temedie (2011), Vandenbergh & Verbrugh (1999), Williams (1963), the nares and the skin of humans and animals may be considered as ecological niche for S. aureus colonization but this colonization does not frequently result into infection thereby tagging the bacterium a normal flora of these body parts. S. aureus colonizes the nares and the skin but if there is an abrasion, lesion or wound in these parts, S. aureus may migrate into the body or blood and cause infections. These infections are called opportunistic (staphylococcal) infections. Compared to other pathogens, S. aureus has a high tendency and proneness to become resistant to antimicrobials (Weese, 2010). This fact, coupled with the constant abuse of drugs and lack of control in the sales of antibiotics contributes to the increasing problem in multidrug resistance of S. aureus including methicillin and vancomycin which is considered as the first line of treatment against methicillin resistant Staphylococcus aureus (MRSA). Cuny et al. (2015), Fluit (2012), Johnson (2011) and Morgan (2008) established that methicillin resistant S. aureus (a multidrug resistant organism) may not only be a nosocomial and community acquired infection but it could also be a zoonotic infection as it can be transmitted from animal to human. The presence of multidrug resistant S. aureus in the nares of cattle poses a threat to cattle herders, butchers, beef retailers/handlers and consumers as these cattle are frequently asymptomatic carriers hence are considered ‘healthy’. This study may evaluate the prevalence of multiple antibiotic resistant livestock associated S. aureus and suggest possible control to diseases caused by multiple antibiotic resistant livestock associated S. aureus.
1.3 Objective of the Study
The general objectives of the study were to evaluate the prevalence of multidrug resistant livestock associated S. aureus and suggest possible control to staphylococcal infections in humans caused by livestock associated S. aureus using the antibiogram of the isolates. The specific objectives are to:
- isolate and identify S. aureus from nasal passage of healthy cattle by Gram staining and biochemical tests (catalase test, slide coagulase test and fermentation of mannitol);
- determine the antibiogram of the isolates and calculate the Multiple Antibiotic Resistance Index (M.A.R.I) using the antibiogram of the isolates;
- determine the minimum inhibitory concentration (μg/disc) of the S. aureus isolates to vancomycin;
- determine the minimum inhibitory concentration (μg/ml) of the isolates to flucloxacillin as a test for methicillin resistant S. aureus (MRSA) and vancomycin and
- determine the synergistic, antagonistic or additive effect of two antibiotics to suggest possible control of staphylococcal infections in human caused by multidrug resistant livestock associated S. aureus.
1.4 Research Questions
- Can aureus beisolated and identified from nasal passage of healthy cattle?
- How is the antibibiogram of aureus isolates determined and how are the Multiple Antibiotic Resistance Index of the isolates calculated using the antibiogram of the isolates?
- How is the minimum inhibitory concentration (μg/disc) of aureus determined?
- How is the minimum inhibitory concentration (μg/ml) of aureus isolated from nasal passage of healthy cattle to flucloxacillin and vancomycin determined?
- Are the effects of the combined antibiotics synergistic, additive or antagonistic?
1.5 Significance of the Study
- This result may create more awareness on the danger of multidrug resistant S. aureus in ruminant flocks.
- This result may educate the general public on the effect of negligence of multidrug resistant S. aureus in cattle and its effect in the consumption of undercooked beef.
- The result may suggest possible control of staphylococcal infections in humans caused by livestock associated S. aureus.
1.6 Justification for the Study
The study may provide a more recent data on multiple antibiotic resistant S. aureus and suggest possible control of livestock associated staphylococcal infections in human.
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