ABSTRACT
Extended-spectrum β-lactamases (ESBLs) are a group of plasmid mediated, diverse complex and rapidly evolving enzymes that are posing a major therapeutic challenge today in the treatment of hospitalized and community based patients.In the presnt study, 10 samples each of the wash water of ready to eat vegetables (tomatoes and cabbage) were collected from different strategic market vendors in Abia state and were analyzed for the screening of Extended Spectrum Beta- Lactamase producing organisms on ready to eat food(wash water of vegetables)tomatoes and cabbage. Organisms isolated were Escherichia coli,Shigella spp, Salmonella spp, Klebsiella spp, using different culture media and biochemical test. At present organizations such as the Clinical and Laboratory Standards Institute provided guidelines for the detection of ESBLs in Escherichia coli, Klebsiella spp, Salmonella spp and Shigella spp in reasy to eat food (wash water of vegetables)tomatoes and cabbage. Using antibiotic multi disk for determining resistance and susceptibility percentage, use of the CLSI breakpoint standard to screen and the use of the Double Disk Synergy Test (DDST) to confirm ESBLs contained in the sample. More effective drugs such as carbapenems, cephamycins, fosfomycin, nitrofurantoin, beta-lactamase inhibitor (clavulanic acid, tazobactam, or sulbactam), colistin should be used more often for treatment of infections.
TABLE OF CONTENTS
Title
Page i
Certification ii
Dedication iii
Acknowledgement iv
Table
of Contents v-vii
List
of Tables viii
Abstract ix
CHAPTER
ONE
1.0 Introduction 1
1.1 Aims
and Objectives 3
CHAPTER
TWO
2.0 Literature
Review 4
2.1 Shelf
Life of Fresh-Cut Fruits and Vegetable 4
2.2 Impact
of Microbiological Spoilage 5
2.3 Sources
of Microbial Contamination 6
2.4 Microbiological
Spoilage Mechanisms in Fresh-Cut Fruit and Vegetable 7
2.5 Prevention
and Control of Microbial Spoilage 9
2.6 Methods for
Detection and Isolation of Organisms That Causes Spoilage on
Vegetable 11
2.7 Microbiological
Spoilage Defects of Fresh-Cut Fruit and Vegetables 12
2.8 Bacterial
Pathogens Associated With Fruit and Vegetable Contamination 13
2.8.1 Campylobacter 13
2.8.2 Pathogenic Escherichia coli 14
2.8.3 Salmonella spp 14
2.8.4 Shigella spp 15
2.8.5 Staphylococcus 15
2.8.6 Vibrio 16
2.8.7 Listeria monocytogenes 17
2.9 ß-Lactamases 17
2.10 Extended
Spectrum Beta-Lactamases 18
2.11 Methods
of Detection Extended Spectrum Beta-Lactamase 19
2.11.1 Screening Tests 19
2.11.2 Confirmatory Tests 19
CHAPTER
THREE
3.0 Materials
and Methods 22
3.1 Materials
and Reagents 22
3.2 Collection
of Samples 22
3.3 Sterilization
of Materials 22
3.4 Preparation
of Culture Media 23
3.5 Isolation
Procedures 23
3.6 Purification
of Isolates 23
3.7 Identification
of the Isolates 24
3.8 Gram
Staining 24
3.9 Biochemical
Test 25
3.9.1 Indole Test 25
3.9.2 Citrate Utilization Test 25
3.9.3 Hydrogen Sulphide (H2S) Production Test 26
3.9.4 Starch Hydrolysis 26
3.9.5 Motility, Indole, Urease (MIU) 26
3.9.6 Coagulase Test 27
3.9.7 Oxidase Test 27
3.10 Antibiotic
Susceptibility Testing 28
3.11 Tests
for Extended Spectrum Beta Lactamase Production 29 3.11.3 Detection of Extended Spectrum Β-
Lactamases Producers 29
CHAPTER
FOUR
4.0 Results 30
4.1 Viable
Microbial Count 30
4.2 Morphology and Biochemical
Identification of Bacteria 31
4.3 Percentage
Occurrence of the Isolated Bacteria 31
4.4 Antibiotics Susceptibility Pattern for
all Isolates 31 4.5 Occurrence of ESBLs among Bacterial Isolates Screened Based on CLSI
Breakpoint 32
4.6
Prevalence of β-Lactamase among the Isolates from the Wash Water of Vegetables using
Double Disk Synergy Test (DDST) 32
CHAPTER
FIVE
5.0 Discussion,
Conclusion and Recommendation 48
5.1 Discussion 48
5.2 Conclusion
50
5.4 Recommendation 51
References 52
Appendix 53
LIST OF TABLES
Table 1: Total
bacterial count of ready to eat food from wash water vegetable of Tomatoes
and Cabbage. 33
Table 2: Morphology
and biochemical, identification of bacteria isolated from ready to eat food (wash water of vegetable, Tomatoes, cabbage) 34
Table 3: Percentage
occurrence of Bacteria isolated from wash water in tomatoes
and cabbage 35
Table 4: Antibiotics
susceptibility pattern for Escherichia
coli isolated from wash
water of Tomatoes. 36
Table 4.1: Antibiotics susceptibility pattern for Escherichia coli isolated from wash water of cabbage. 37
Table 4.2: Antibiotics
susceptibility pattern of Shigella spp isolated from wash water
of Tomatoes. 38
Table 4.3: Antibiotics
susceptibility pattern of Shigella spp
isolated from wash water
of cabbage 39
Table 4.4: Antibiotics susceptibility pattern of Salmonella spp isolated from wash water
of Tomatoes. 40
Table 4.5: Antibiotics susceptibility pattern of Salmonella spp isolated from wash water
of Cabbage. 41
Table 4.6: Antibiotics susceptibility pattern of Klebsiella spp isolated from wash water
of Tomato 42
Table 4,7: Antibiotics susceptibility pattern of Klebsiella spp isolated from wash water
of Cabbage. 43
Table 5: Antibiotics susceptibility and Resistance pattern of all
isolates from wash water
of Tomatoes. 44
Table 5.1: Antibiotics susceptibility and Resistance pattern of all
isolates from wash water
of cabbage. 45
Table 6: Occurrence of ESBLs among bacterial isolate screened based
on the CLSI
breakpoints 46
Table 7: Confirmation of ESBL among the bacteria isolates based on
Double Disks
Synergy Test (DDST) 47
CHAPTER ONE
1.0 INTRODUCTION
Vegetable is a very common food accompaniment in Nigeria.
The vegetables that usually make up this recipe include tomatoes, cucumber,
carrots, cabbage and lettuce. They are sold in almost every market, and can be
seen hawked around by traders. Fruits and vegetable have been identified as
significant sources of pathogens and chemical contaminants (Uzeh et al., 2009). As a result,
environmental and food microbiologists have continued to identify and suggest
control measures for hazards at all stages in the supply chain. Khan et al. (1992) reported that bacterial
contamination results from various unsanitary cultivation and marketing
practices, In another study, Tambekar et al.
(2006) reported that bacterial contamination of salad vegetable wa`s linked
to the fact that they are usually consumed without any heat treatment. These
vegetables can become contaminated with pathogenic microorganisms during
harvesting, through human handling, harvesting equipments, transport containers,
wild and domestic animals pathogens from the human and animal reservoir as well
as other environmental pathogens can be found at the time of consumption.
Although spoilage bacteria, yeast and mould dominate the micro flora on row
fruits and vegetable, the occasional presence of pathogenic bacteria, parasites
and viruses capable of causing human infection has also been documented (Hassan
et al., 2006). Coliforms are
facultative anaerobic Gram negative rods belonging to the family Enterobacteriacaea. They are known
contaminants of food and water, causing various intestinal and extra-intestinal
infections such as urinary, central nervous system and respiratory tract
infections. The presence of E. coli in
some green leafy vegetables few studies have examined the presence of
entophytes or surface associated bacteria from the perspective of human
consumption, by sampling minimally processed vegetables such as ready-to-eat
salad produce. Similarly, few studies have focused on the entire entophyte
community, rather than just potential pathogens, even though native entophyte
bacterial populations could potentially serve as competitors to such organisms.
However, in Nigeria, local utilization of carrots, cabbage,
onions, and cucumbers is limited to direct unprocessed eating either wholly or
a growing awareness on the need to evaluate microorganism associated with
spoilage of these vegetables.
Beta-lactamases (BLs) has emerged as an important mechanism
of resistance in Gram-Negative bacteria. Β -lactam antibiotics are among the
safest and most frequently prescribed antimicrobial agents all over the world
in treating Gram positive and Gram negative infections. Production of
β-lactamases is the most common mechanism of the bacterial resistant for these
antibiotics. These enzymes are numerous and are plasmid mediated, capable of
hydrolyzing and inactivating a wide variety of β-lactam antibiotics. In
addition, Beta-lactamases producing organisms exhibit co-resistance to many
other classes of antibiotics resulting in limitation of therapeutic option. For
this reason, the significance of such Beta-lactamases–mediated infections has
been increasingl y reported worldwide (Khanfar et al., 2009). This study was
done to determine the Beta-lactamases producing bacteria from burn wound
isolates in our setting and describe their resistance patterns, which would
enable the determination of empirical antibiotic strategies for the early
treatment of imminent septic events.
Extended spectrum beta-lactamase (ESBL) capable of
hydrolyzing penicillins, broad spectrum cephalosporins and monobactams in
Enterobacteriaceae (Paterson and Bonomo, 2005) are often located on plasmids
that are transferable from strain to strain and between bacterial species (Rupp
and Fey, 2003). Extended spectrum beta-lactamase (ESBL) producers have
continued to draw attention globally with their attendant clinical failure to
new generation antibiotics and nosocomial spread (Olowe et al., 2012). In addition, rapid changing over time in extended
spectrum beta-lactamase has been observed with variations within geographic
areas. Clinical outcomes data indicate that extended spectrum beta-lactamase
are clinically significant and when detected, suggest the need for the use of
appropriate antibacterial agents.
1.1 AIMS AND OBJECTIVES
The aim of this research work is to isolate the
microorganisms associated with vegetable wash water and to determine the
occurrence of Beta-lactamases activities among the isolates
The objectives are;
•
To isolate and identify the bacterial isolates present in
the vegetable wash waster samples.
•
To determine the percentage occurrence of the isolates in
the vegetable wash water samples.
•
To determine the antimicrobial susceptibility profile of the
isolate from the vegetable wash waster samples.
•
To screen the isolates for extended spectrum beta-lactamases
production.
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