TABLE OF CONTENTS
TITLE PAGE
CERTIFICATION
DEDICATION
ACKNOWLEDGEMENT
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF PLATES
ABSTRACT
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1Introduction
1.1.1 Aims of the Study
1.1.2 Objectives of the Study
1.1.3Null hypothesis
1.1.4Alternative hypothesis
1.2 Literature Review
1.2.1Antibiotics and their usage
1.2.2 History of Antibiotics
1.2.3Classes of antibiotics
1.2.4β- Lactam antibiotics
1.2.5Mechanisms of Action of Antibiotics
1.2.6.0 Antimicrobial Drug Resistance
1.2.6.1 Antibiotics Resistance: Meaning and History
1.2.6.2 Multidrug Resistant organisms
1.2.7 Causes of Antibiotic Resistance
1.2.7.1Beta- Lactamases
1.2.7.2 Extended spectrum β-Lactamases
1.2 .8 Bacterial Mechanism of Antibiotic resistance
1.2.9 Acquisition and spread of antibiotic resistance in bacteria
1.2.9.1 Inherent (Natural) resistance
1.2.9.2 Acquired Resistance
1.2.9.3 Vertical gene transfer
1.2.9.4Horizontal gene transfer
1.2.10Epidemiology of Resistance
1.2.11 Impact of resistance on public health and Economy
1.2.12 Plasmid extraction and its principle
1.2.13 Plasmid Transfer
1.2.13.1 Conjugation
1.2.13.2 Mechanism of Conjugation: Formation of mating pair
1.2.14 Plasmid Curing
CHAPTER TWO: MATERIALS AND METHODS
2.1 Study area and study population
2.1.1 Sample collection
2.2 Preparation of media for Identification and differentiation of Salmonella and Shigella
species
2.2.1 Isolation and Identification of Salmonella and Shigella species from Human faecal
samples
2.2.3 Gram staining
2.2.4 Urease production
2.2.5 Catalase test
2.2.6 Indole production
2.2.7 Triple Sugar Iron Agar test
2.2.8 Motility test using Motility Indole Urea
2.2.9 Antibiotic Susceptibility Testing
2.2.10Determination of Multiple Antibiotic resistance indices
2.2.11 Plasmid DNAIsolation and profiling
2.2.111. Isolation protocols
2.2.11.2 Agarose Gel electrophoresis (AGE) of plasmid DNA
2.2.12 Plasmid DNA Curing
2.2.12.1 Sodium Dodecyl Sulphate (SDS)
2.2.12.2 Use of Acridine Orange
2.2.13Plasmid Transfer
2.2.14Tests for β-Lactamase production
2.2.14.1 Nitrocefin Test Procedure
2.2.14.2Iodometric test using paper strip method
2.2.14.3Acidimetric test using paper strip method
2.2.15Extended spectrum β-Lactamase via double disc diffusion synergy test
2.3 Statistical Analysis
CHAPTER THREE: RESULTS
3.1 Distribution of Salmonella and Shigella species among male and female patients of
different age groups
3.2 Distribution and Antibiotic Resistance among Salmonella and Shigellaspecies in different Human age groups
3.3 Distribution of Antibiotic Resistance among Salmonella and Shigella species isolated from Human faecal Samples
3.4 Plasmid DNA distributions among Salmonellaand Shigella isolates
3.5 Effect of Plasmid Curing on the Resistance Pattern of some Multidrug Resistant (MDR) isolates
3.6 Conjugative Transfer of Multidrug Resistant among species of Salmonella, Shigella and E. coliisolated from Human faecal Samples
3.7 Beta-Lactamase production
3.8 Extended spectrum β-Lactamase in some Salmonella and Shigella isolates
3.9 Statistical deduction on production of Beta-lactamase
3.10 Statistical deduction from Antibiotic Susceptibility test results
CHAPTER FOUR
4.1 Discussion
4.2 Conclusion
REFERENCES
APPENDIX 1: LABORATORY MEDIA
APPENDIX 11: KEY REAGENTS
APPENDIX: STATISTICAL TABLES
ABSTRACT
Salmonella and Shigella species isolated from human faecal samples were examined for Beta-lactamase production and resistance to some antibiotic agents. Age distribution of sources of resistant isolates showed that Shigellaisolates from Youths (18 - 30 years) were more resistant to Ampicillin (66.67%) and Augmentin (33.33%) than isolates from Infants (6 months - 4 years) which showed percentage resistance of 3% to Ampicillin and 0% to Augmentin.Salmonella species isolated from Adults (31 – above) were more resistant to Augmentin (45%) and Ampicillin (40%), than isolates from Youths (18- 31 years) which showed percentage resistance of 40% to Ampicillin and 36.67% to Augmentin. Salmonella isolated from Infants (6 months – 4 years) showed p ercentage resistance of 28.57% to Ampicillin and 14.29% to Augmentin while isolates from Children (5 - 17 years) showed percentage resistance of 25% to Ampicillin and 12.5% to Augmentin. Sex distribution of sources of resistant isolates showed that Shigella species isolated from males were more resistant to Ampicillin (100%) while isolates from Females were more resistant to Augmentin (50%). Salmonella species isolated from Males showed high percentage resistance to Augmentin (60%) while isolates from Females were more resistant to Ampicillin (57.14%).
Salmonella species showed higher percentage resistance to commonly used antibiotic agents than Shigella species. The result showed that 8% of Shigellaspecies and 21.5% of
Salmonellaspecies were resistant to more than eight antibiotics with multiple antibiotic resistance (MAR) index ranging from 0.2-0.9. Presumptive results of resistance curing treatments showed that the resistance traits were plasmid borne. Agarose gel electrophoresis (AGE) showed plasmids with molecular weights clustered around 23.1kb for species of
Salmonella and Shigella. Conjugative transfer of resistance determinants was demonstrated from Salmonella to E. coli and from Salmonella to Shigella but not from Shigella to E. coli or to Salmonella. Studies on Beta-lactamase production showed that 9 (81.82%) of Shigella species and 22 (61.11%) ofSalmonella species were Beta-lactamase producers with 3 (27.27%) of Shigellaspecies and 20 (55.56%) of Salmonella species producing extended spectrum Beta-lactamases (ESBLs). ESBLs production is an acknowledge threat to modern medicine which is antibiotic based.
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
In human medicine, the most important family of bacteria is Enterobacteriaceae, which includes genera and species that cause well-defined diseases, as well as nosocomial infections. The members of this family are Gram-negative, rod-shaped, non-spore-forming facultative anaerobes that ferment glucose and other sugars, reduce nitrate to nitrite, and produce catalase but seldom oxidase. Most Enterobacteriaceae are components of the gastrointestinal flora of humans and animals, although many are also widespread in the environment. Furthermore, these bacteria can cause many different infections, such as septicaemia, urinary tract infections, pneumonia, cholecystitis, cholangitis, peritonitis, wound infections, meningitis, and gastroenteritis, and they can give rise to sporadic infections or outbreaks (Donnenberg, 2009).
Salmonella and Shigella infections represent a major health problem worldwide, particularly in developing countries where they are recognized as the most frequent causes of morbidity and mortality (David and Frank, 2000, Mahbubur et al., 2007; Abdel et al., 2008). Life lost, together with the high costs to local public health care system, makes prevention and control a priority (Mahbubur et al., 2007; Yah et al., 2007a). The two pathogens have been associated with diarrhoea but the severity of the diarrhoea varies with the pathogens. Generally Shigella causes bloody diarrhoea while Salmonellainduces non-bloody gastroenteritis. Antibiotic resistant Salmonella and Shigellaare of global concern because they affect both developed and developing countries due to increased international travel (David and Frank, 2000, Duboiset al., 2007).These concerns have been further reinforced in recent years by the emergence of antimicrobial resistance among major groups of the enteric pathogens. The presence of antibiotic resistant bacteria from hospitalized patients throughout the world has been documented (Yah et al., 2007b).
Studies with Salmonella and Shigella are of particular relevance because these species can occupy multiple niches, including human and animal hosts (Martinet al., 1996, Levy, 1998; Khan, 2006). Reports have shown that the resistance of gastroenteric Salmonella and Shigella strains to antimicrobial agents is in large part due to the production of extended- spectrum β-lactamases (ESBLs) encoded on plasmids, as well as on the chromosome (David and Frank 2000). In Gram-negative pathogens, β-lactamases remain the most important contributing
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