ABSTRACT
Screening of kunun-zaki samples for Methicillin Resistant Staphylococcus aureus, Vancomycin Resistant Staphylococcus aureus and Extended Spectrum Beta Lactamase producing Salmonella was investigated. A total of 40 samples were collected and a total of 19 Salmonella and 22 Staphylococcus aureus were isolated. The total Salmonella count ranged from 3.4 x 105 to 1.90 x 106 with a mean of 14.2 x 105 while the S.aureus count ranged from 3.4 x 105 to 1.58 x 106 with a mean of 184.5 x 105. All the isolated organisms were subjected to in-vitro antibiotic susceptibility test using Kirby Bauer disc diffusion method. A total of 14 antibiotic discs namely Ceftazidime (CAZ 30μg), Cefuroxime (CRX 30μg), Gentamicin (GEN 10μg), Ciprofloxacin (CPR 5μg), Ofloxacin (OFL 5μg), Augumentin (AUG 30μg), Nitrofurantoin (NIT 300μg), Ampicillin (AMP 10μg), Imipenem (IMP 10μg), Erythromycin (E 30μg), Meropenem (MER 10μg), Vancomycin (VAN 10μg), Cloxicillin (CXC 5μg), and Oxacillin (OX 10μg) were used. Most of the Salmonella isolates were susceptible to Ceftazidime (94.7%) followed by Ofloxacin (89.5%) while they exhibited resistance to Meropenem (73.7%) and Imipenem (57.9%). Also the Salmonella isolates were screened for ESBL production in accordance to the CLSI breakpoint and 12(63.2) were positive. On subjection of the 12 isolates to double disc synergy test (DDST), 5 (41.7%) were confirmed to be ESBL producers. A good number of the S.aureus isolates were susceptible to Ofloxacin (95.5%) followed by Augumentin (72.7%) and when subjected to MRSA testing using Oxacillin, 9(40.9%) were resistant while 10(45.5%) and 3(13.6%) exhibited intermediate and susceptible results to Oxacillin respectively. VRSA were also confirmed when the isolates were subjected to Vancomycin antibiotic and 5(22.7%) isolates were resistant while 15(68.2%) and 2(9.1%) had intermediate and susceptible results respectively.
TABLE
OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of Contents v
List of Tables ix
List of figures x
Abstract xi
CHAPTER
ONE
INTRODUCTION 1
1.1
Introduction 1
1.2
Rationale 1
1.3
Objectives
of the study 4
CHAPTER
TWO
LITERATURE REVIEW 5
2.1
Production
and preservation of kunun-zaki 5
2.2
Nutritive
value of kunun-zaki 9
2.3
Salmonella:
A major contaminant of kunun-zaki 9
2.3.1
Description
of the organism 9
2.3.2
Growth
and survival characteristics 11
2.3.3
Symptoms
of disease 12
2.3.4
Virulence
and infectivity 13
2.3.5
Epidemiology
of Salmonella 15
2.3.6
Clinical
syndromes 17
2.3.6.1 The “Enteric fevers”
(Typhoid fever) 17
2.3.6.2 Bacteremia with focal
lesions 18
2.3.6.3 Enterocolitis 19
2.3.7 Detection of Salmonella 20
2.3.8 Occurrence in food 20
2.3.9 Antibiotic considerations
and multi-drug resistance in Salmonella 21
2.4 Staphylococcus aureus, A major Contaminant of kunun-zaki 24
2.4.1 Genome characteristics 24
2.4.2 Natural habitats and
reservoirs of Staphylococcus species 24
2.4.3 Pathogenic members of the
genus Staphylococcus 25
2.4.4 General characteristics
and history of Staphylococcus aureus 25
2.4.5 Epidemiology and
transmission of S.aureus 27
2.4.6 Virulence factors of S.aureus 28
2.4.6.1 Pyrogenic toxin super antigens 28
2.4.6.2 Exoliative toxins 28
2.4.6.3 Other toxins 29
2.4.6.4 Role of pigment in
Virulence 29
2.4.7 Treatment and Antibiotic
resistance 29
2.4.8 Mechanism of antibiotic
resistance 30
2.5 Methicillin – Resistant Staphylococcus aureus (MRSA) organisms 32
2.5.1 Community- associated MRSA
(CA-MRSA) in humans 33
2.5.2 Hospital- associated MRSA
(HA-MRSA) in humans 33
2.5.3 Resistance and virulence
factors associated with MRSA 34
2.5.4 Transmission of MRSA 35
2.5.4.1 Transmission of MRSA in
Humans 35
2.5.4.2 Zoonotic nature of MRSA and transmission in animals 36
2.5.5 Treatment
of MRSA in Humans 37
2.6 VRSA
and VISA 38
2.7 Antibiotics 39
2.7.1 Antibiotics
that inhibit cell wall synthesis 40
2.7.2 Antibiotics
that disrupt cell membrane function 41
2.7.3 Antibiotics
that inhibit protein synthesis 41
2.7.4 Antibiotics
that inhibit nuclei acid synthesis 42
2.7.5 Antibiotics
that act as antimetabolites 43
2.8 Beta-Lactam
antibiotics 45
2.8.1 Penicillin 45
2.8.2 Cephalosporins 47
2.8.3 Monobactams 50
2.8.4 Carbapenems 50
2.9 Antimicrobial Resistance 51
2.9.1 Beta Lactamase 55
2.9.2 Extended spectrum
Beta-Lactamases 56
CHAPTER
THREE
MATERIALS AND METHODS 60
3.1 Study area 60
3.2 Collection of Samples 60
3.3 Sterilization of
Materials 61
3.4 Media used and their
preparation 61
3.5 Microbiological analysis
of samples 61
3.5.1 Serial Dilution 61
3.5.2 Isolation and Enumeration 62
3.5.3 Inoculation of Salmonella
spp 62
3.6 Identification of
Bacterial Isolates 63
3.6.1 Purification and storage
of the isolates 63
3.6.2 Isolation and Identification
of bacteria isolates 63
3.6.2.1 Gram staining 63
3.6.2.2 Biochemical Identification
of Bacterial Isolates 64
3.6.2.2.1Catalase test 64
3.6.2.2.2Coagulase test 65
3.6.2.2.3Citrate test 65
3.6.2.2.4 Oxidase test 65
3.6.2.2.5Indole Test 66
3.6.2.2.6Triple Sugar Iron (TSI) test 66
3.6.2.2.7 Motility Test 67
3.6.2.2.8 Urease Test 67
3.7 Standardization of
inoculum 67
3.8 Antibiotic Susceptibility
Test 68
3.9 Clinical Laboratory Standard Institute (CLSI) Breakpoint Test for
ESBLs
Screening 69
3.10 Double Disc Synergy Test (DDST) for ESBL confirmation 69
CHAPTER FOUR
RESULTS 71
4.0 Results 72
CHAPTER FIVE
DISCUSSION, RECOMMENDATIONS AND CONCLUSION 85
5.1 Discussion 85
5.2 Conclusion 88
5.3 Recommendations 89
References 90
LIST OF FIGURES
Figure Title Page
Figure 2.1 Flow diagram of traditional kunun-zaki
production 8
LIST OF TABLES
Table Title Page
4.1
Incidence
and percentage occurrence of the Salmonella and S.aureus Isolates
In the kunun-zaki samples 74
4.2
The Salmonella count (cfu/mg) of the
kunun-zaki samples 75
4.3
The S.aureus count (cfu/mg) of the
kunun-zaki samples 76
4.4
Antibiotic
susceptibility pattern of the isolated Salmonella
isolates from the
Kunun-zaki samples 77
4.5
Zones of
inhibition for the screening for ESBLs based on CLSI breakpoint using
Ceftriaxone (CTR), Ceftadime (CAZ) and
Aztreonam (ATM). 78
4.6
Confirmation of ESBLs among the Salmonella isolates based on the Double
Disc Synergy Test. (DDST) 79
4.7
Antibiotic susceptibility pattern of the isolated S.aureus isolates from the kunun-zaki
samples. 80
4.8
Prevalence of MRSA among the S.aureus
isolates from the kunun-zaki samples based
on resistance to Oxacillin antibiotic discs 81
4.9 Prevalence of VRSA among the S.aureus
isolates from the kunun-zaki samples 82
4.10 Cultural, morphological and biochemical
characteristic of the Salmonella and
S.aureus isolates 83
CHAPTER ONE
INTRODUCTION
1.1 Introduction
Foods and drinks are
substances ingested by all living organisms for the continuance of life.
However, foods consumed by humans and animals serve as favourable media for
rapid growth of microorganisms because of the abundant organic matter, moisture
and suitable pH contained and because of the fact that the microorganisms are
equally living organisms. Presence of these microorganisms in foods and drinks
warrants their rapid multiplication which renders the food unfit and dangerous for
human consumption.
Foods and drinks
commercialized in markets have likelihood of being exposed to pathogenic
microorganisms because of poor handling by the sellers. Waterborne and other
related pathogens including bacteria are spread in water either through human
ingestion of contaminated water or drinks. Meanwhile, faecal pathogens present
in contaminated water used in the production of some drinks are due to
ineffective sanitary measures. The pathogens as a result, get into the locally
made drinks such as millet juice (Kunun-zaki) which require water as a growth
medium and ingredient.
1.2 Rationale
Kunun-zaki (Hausa) is an indigenous
fermented non-alcoholic, non-carbonated and refreshing cereal beverage popular beverage.
It is a consumed by large population of people in northern Nigeria. Preparation
protocol varies amongst people and can generally be produced from either the
following substrates; millet (Pennisetum typhoideum), maize (Zea mays)
or sorghum (Sorghum bicolor), but millet is the most common substrates
(Akoma et al. 2006). Spices such as ginger, black pepper, red pepper,
cloves and sweeteners such honey and sugar together with small quantities of
sweet potatoes sugar are commonly added as flavor and taste improver (Ahmed et
al. 2003). The drink is very cheap because the cereals and additives used
in its production are locally sourced as they are grown throughout the savannah
belt of West Africa. The drink is produced from fermented millet, sorghum,
guinea-corn and maize in decreasing order of preference.
In some cultures, the grains are used in a composite form especially
millet, guinea-corn and sorghum in a ratio of 1:2 w/w (Abegaz, 2007). Because
these are agricultural commodities, they may contain a high level of microbial
impurities (Adeyemi and Umar, 1994; Bibek, 2001). Kunun-zaki is
rich in carbohydrates, vitamins and minerals but low in proteins.
It is generally consumed on its own by adults,
as a thirst quencher or serves as refreshment in some communities, used for
entertainment at homes and during unique ceremonies like Christmas and Sallah,
and sometimes it is used as a weaning drink for infants (Gaffa, 2000 Elmamood
and Doughari, 2007; Okechukwu et al., 2011). Non-alcoholic nature of
this drink made it to be readily consumed by both Muslims and Christians alike
(Okechukwu et al., 2011).
The production methods are crude, ingredient concentrations are
neither quantified nor standardized, and instead preparation is largely a
matter of family tradition (Onuorah, et al., 1987). Significant
variation exists in the production procedure depending on the cultural habits
of its consumers leading to differences in quality and stability (Aboh and
Oladosu, 2014).
The high water content (about 85%) coupled with crude methods of
production and packaging under inadequate sanitary conditions predisposes Kunun-zaki to microbial
contamination (Elmahmood et al., 2007). The ubiquitous
nature of microorganism guarantees them the opportunity to be found in this
locally made beverage drink and possibly in the water used for its preparation,
during storage and other processes involved in its preparation. Some of the
bacterial species that are associated with this locally produced beverage drink
include spoilage species, pathogenic species, coliforms and lactic acid
bacteria (Amusa and Ashaye, 2009; Bukar et al., 2010; Akoma et al.,
2012).
The occurrence of wide genera of microorganisms could be
attributed to the unhygienic conditions of preparation and the use of
contaminated raw materials and utensils (Ayo,
2004). The
presence of bacteria such as Staphylococcus
aureus, Escherichia coli and Salmonella
spp. could be a serious problem as Staphylococcus
aureus is a normal flora of the skin, palms,
nose, throat, mucus membrane and a common ecological
agent of septic arthritis (Willey et al., 2008).
Escherichia coli is an important member of the coliform group. It is a part of the normal flora of the
human intestine and some vertebrates, even
though strain of this organism can be
pathogenic. The presence of these pathogens
even in small numbers could render a beverage
unsuitable for human consumption (Amusa and Ashaye,
2009; Essien et al., 2009; Lawal, 2012).
It is very possible that the contamination by these pathogens
could have occurred, during hawking and packaging
of the products due to improper or careless handling
of the products leading to contamination of products.
Food contamination by specific organisms is largely
due to the presence of these organisms and their entrance
into the food or beverage drinks as a result of poor hygiene
and sanitation (Bukar et al., 2010).
The micro flora of finished product depends on the processing and
storage conditions. The pH of Kunun-zaki
is usually too low to allow the growth of pathogenic microorganisms
but the presence of Staphylococcus aureus and other coliforms such as Salmonella
could be a matter of serious concern.
Packaging
materials are also cheap and easily available. It is usually packaged and sold
in 1litre and 500 ml plastic bottles or even tied in small polyethene bags. Kunun-zaki is also not shelf-stable, readily undergoing microbial
induced spoilage within 2-3 days hence it must be consumed within
18 - 36 h of production due to its poor keeping quality.
The
high water content coupled with crude methods of production and packaging under
improper sanitary conditions predisposes Kunun-zaki
to microbial contamination which could resistant to known
antibiotics. This study was designed to screen Kunun-zaki for Methicillin Resistant Staphylococcus aureus
(MRSA), Vancomycin Resistant Staphylococcus aureus (VRSA) and Extended
Spectrum Beta Lactamase (ESBL) producing Salmonella. spp which are microbial food contaminants of Kunun-zaki.
1.3
Objectives
1. To
isolate and identify Staphylococcus
aureus and Salmonella in
Kunun-zaki.
2. To
determine the load of Staphylococcus
aureus and Salmonella in Kunun-zaki.
3. To
determine the antibiotic susceptibility pattern of Staphylococcus aureus and Salmonella
isolates to different classes of antibiotics.
4. To
detect the presence of MRSA, VRSA and ESBL producing Salmonella.
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