BINDURA UNIVERSITY OF SCIENCE EDUCATION
EDUCATION DEPARTMENT OF CURRICULUM STUDIES
DETERMINATION OF THE IN-VITRO ANTIBACTERIAL PROPERTIES OF ALLIUM CEPA EXTRACTS ON STAPHYLOCOCCUS EPIDERMIDIS AND ESCHERICHIA COLI.
By
IRENE MUYENGWA (B1231805)
A PROJECT SUBMITTED IN PARTIAL FULFILMENT OF THE BACHELOR OF SCIENCE EDUCATION HONOURS IN BIOLOGICAL SCIENCES
MAY 2016
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DECLARATION
I Irene Muyengwa declare that this project for Bachelor of Science Education Honours in
Biological Sciences is my original work and has not been previously submitted to this or any other university.
Signed…………………………………. Date…………………………………………………
Witness…………………………………Date………………………………………………….
SUPERVISOR
I ……………………………………………… declare that I have supervised this project and I am satisfied that it can be submitted to the Faculty of Science Education of Bindura University of Science Education.
Date…………………………..
Signature…………………….
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ABSTRACT
Antibacterial activities of Allium cepa against Staphylococcus epidermidis and Escherichia coli were determined using disk diffusion and tube dilution tests. Penicillin G sodium salt was used as the positive control. A. cepa had antibacterial activity against S.epidermidis
(mean MIC 0.096, ±0.11 and mean MBC 1.75, ±0) and E. coli (mean inhibition diameter
26.5 ± 6.02). Penicillin G sodium salt was a stronger antibacterial agent than the A. cepa herb. Due to an increase in bacterial resistance and the inability to access antibiotics, natural, herbal plants like A. cepa can be used in prevention and treatment. The plant A. cepa had antimicrobial effects and therefore can be used to achieve therapeutic effects. However, there is need to study on whether A. cepa has antagonistic or synergistic effects.
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DEDICATION
To my husband Gift and sons Kelvin, Bright and Mukudzeishe.
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TABLE OF CONTENTS PAGE
Declaration ...... i
Release form ...... ii
Approval form ...... iii
Dedication ...... iv
Acknowledgements ...... v
Abstract ...... vi
Table of contents ...... vii
List of appendices ...... xii
List of tables ...... xiii
List of figures ...... xiv
1.0 INTRODUCTION ...... 1
1.1 General Introduction ...... 1
1.2 Background to study ...... 2
1.3 Statement of problem...... 4
1.4 Justification ...... 4
1.5 Aims and objectives ...... 5
1.5.1 Aims ...... 5
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1.5.2 Objectives ...... 5
1.6 Assumptions ...... 5
1.7 Limitations ...... 5
1.8 Delimitations ...... 6
2.0 LITERATURE REVIEW ...... 7
2.1 Plant description ...... 7
2.2 Nutritional content of A. cepa ...... 7
2.3 Medicinal uses ...... 9
2.4 Antimicrobial properties of A. cepa ...... 10
2.4.1 Antibacterial effects...... 10
2.4.2 Antifungal effects ...... 10
2.4.3 Antimicrobial properties of raw and cooked onion ...... 11
2.5 Test organisms used in research ...... 11
2.5.1 Staphylococcus epidermidis description ...... 11
2.5.2 Escherichia coli description ...... 12
2.6 Antibacterial agents ...... 13
2.6.1 Characteristics of antibacterial agents ...... 13
2.6.2 Media recommended for susceptibility testing ...... 14
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2.6.3 Methods used to determine susceptibility of bacteria to anti bacteria agents ...... 14
2.6.3.1 McFarland turbidity standard ...... 15
2.6.3.2 Kirby-Bauer method ...... 15
2.6.3.3 Tube dilution method ...... 16
3.0 RESEARCH METHODOLOGY ...... 18
3.1 Study site and sample collection ...... 18
3.2 Preparation of A. cepa extract ...... 18
3.3 Preparation of media and solutions ...... 18
3.3.1 Mannitol salt agar ...... 18
3.3.2 Nutrient agar ...... 19
3.3.3 Nutrient broth...... 19
3.3.4 Preparation of penicillin G sodium solution ...... 19
3.4 Preparation and maintenance of test organisms ...... 19
3.4.1 Staphylococcus epidermidis ...... 19
3.4.2 Escherichia coli ...... 20
3.5 Identification of cultures used as test organisms ...... 20
3.5.1 Staphylococcus epidermidis ...... 20
3.5.1.1 Growth on mannitol salt agar ...... 20
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3.5.1.2 Gram stain ...... 20
3.5.2 Escherichia coli ...... 21
3.5.2.1 Growth on nutrient agar ...... 21
3.5.2.2 Gram stain ...... 21
3.6 Preparation and maintenance of stock culture ...... 22
3.6.1 Staphylococcus epidermidis ...... 22
3.6.2 Escherichia coli ...... 22
3.7 Antibacterial test ...... 22
3.7.1 McFarland turbidity standard preparation ...... 22
3.7.2 Use of 0,5 McFarland standard ...... 23
3.7.3 Disk diffusion test ...... 23
3.7.3.1 Preparation of disks ...... 23
3.7.3.2 Susceptibility test for Staphylococcus epidermidis ...... 23
3.7.3.3 Susceptibility for Escherichia coli ...... 24
3.7.4 Tube dilution test ...... 24
3.7.4.1 Determination of minimum inhibitory concentration ...... 25
3.7.4.2 Determination of minimum bactericidal concentration ...... 25
4.0 RESULTS ...... 27
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4.1 Identification of bacterial cultures ...... 27
4.1.1 Staphylococcus epidermidis ...... 27
4.1.2 Escherichia coli ...... 27
4.2 0.5 McFarland turbidity standard ...... 27
4.2.1 Disk diffusion test ...... 28
4.2.1.1 Staphylococcus epidermidis ...... 28
4.2.1.2 Escherichia coli ...... 30
4.2.2 Tube dilution test ...... 32
5.0 DISCUSSION AND CONCLUSION ...... 33
5.1 Discussion ...... 33
5.2 Conclusion ...... 36
REFERENCES ...... 37
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CHAPTER 1: INTRODUCTION
1:1 GENERAL INTRODUCTION
Allium cepa, (bulb onion) is a familiar and widely cultivated vegetable of the genus Allium, and family Alliaceae (Yang et al., 2004). In Shona and Ndebele, the vegetable is called hanyanisi and ithanga respectively. It is usually served cooked or raw. When chopped, onions are pungent as they contain chemical compounds which irritate the eyes.
Traditionally onions and other plants belonging to the Allium genus have been used as herbal remedy for common cold, heart disease, diabetes and sore throat, (Rose et al., 2005). In
Zimbabwe’s folk medicine, a large onion bulb is cut in cross section, sprinkled with brown sugar and inverted to let the juice out. This is given to one with flu and cough. When put on pillow overnight, bulb onion peels are said to reduce nasal blockages caused by flu. When applied regularly to newly forming acne, bulb onion extract reduces redness, inflammation and eventually size of scar (Verbal communication). However, bulb onion has been identified to be toxic to dogs, cats, guinea pigs and monkeys due to sulfoxides which these animals are unable to digest (Cope, 2005; Salgado et al., 2011)
In vitro studies have shown that infectious bacterial and fungal species like Salmonella typhi and Candida albicans respectively are susceptible to A. cepa extract (Bakht et al., 2013).
Staphylococcus epidermidis is a spherical gram positive bacterium of the genus
Staphylococcus. Staphylococcus genus has at least forty species most of which are harmless.
However, a species can have both harmless and harmful strains. For example, Staphylococcus aureus are commensals of the human skin flora while its harmful strains cause skin infections like boils, respiratory diseases like sinusitis and food poisoning (Levinson, 2010).
Staphylococcus saprophyticus is part of the normal vaginal flora.
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S. epidermidis species are frequently found on the skin and in the human respiratory tract (Fey and Olson, 2010).Though not always pathogenic, S. epidermidis is a frequent contaminant of specimens sent to diagnostic labs and also infects patients with compromised immune systems
(Levinson, 2010). Of particular concern is its formation of biofilms on catheters and surgical implants thus causing post-operative wound infections (Otto, 2009).S. epidermidis is resistant to heat and high salt concentrations and can survive long periods on dry inanimate objects.
These characteristics of S. epidermidis make it persistent in nature. On skin surfaces S. epidermidis is often found because the bacteria can tolerate the low moisture and high salt content (Menichetti, 2005). It can easily spread from person to person via hand to hand contact and can penetrate the deep tissues of skin damaged by burns, cuts, and skin conditions like acne and eczema (Levinson, 2010).
Escherichia coli are rod shaped, gram negative bacteria commonly found in the lower intestines of warm blooded animals. It provides a microbial derived vitamin K for the host and prevents colonisation of gut by pathogenic bacteria (Hudault et al., 2001). Though having commensal relationships with hosts, a number of species of the genus Escherichia are pathogenic. For example Escherichia fergusonii is known to colonise open wounds of both humans and animals and also cause urinary tract infections (Mahapatra et al., 2005). Virulent E. coli strains are a common cause of gastro-intestinal diseases ranging from simple diarrhoea to dysentery and urinary tract infections (Hudault et al., 2001).
High prevalence of bacterial infections, development of antibiotic resistance and high cost of antimicrobials has become a global public health challenge leading to increase in diseases and death rate.
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1.2: BACKGROUND TO STUDY
Pathogenic S. epidermidis strains are a frequent cause of clinically important nosocomial infections at healthcare institutions. E. coli is a major cause of foodborne illnesses characterised by abdominal pains, diarrhoea and sometimes fever (Mahapatra et al., 2005). This largely contributes to the growing economic burden for treatment of these infections which are among the major causes of death among patients.
In a study to identify the important bacterial pathogens responsible for wound infections secondary to snakebite, which is a serious and important problem in tropical and subtropical countries, S. aureus was the most common isolate followed by Escherichia coli (Garg et al.,
2009). In Southern Africa, malnourished children and residents of long term care facilities have highest risk of methicillin-resistant S. aureus (MRSA) (Naidoo et al., 2013). In Zimbabwe, hospital acquired S. epidermidis infection is of particular concern. Chingarande and Chidakwa
(2013) identified S. epidermidis as one of the many isolates from radiology equipment swabs hence causing post-operative wound infections.
E. coli bacterial infections are rampant especially in Zimbabwean towns due to improper sewer and waste disposal. This has led to an increased environmental contamination and thus infection of individuals consuming contaminated water and food.
Due to the prevailing economic challenges visiting doctors for prescription and treatment of E. coli and S. epidermidis bacterial infection is beyond the reach of many in Zimbabwe. In the event that antibiotics have been found, patients are not compliant thus causing development of resistant bacterial strains. The rise in incidence has been accompanied by a rise in antibiotic resistant strains specifically methicillin resistant S. epidermidis strains and the recently discovered vancomycin resistant enterococci strains (Chingarande and Chidhakwa, 2013).
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Antimicrobial resistance requires new drug inventions thus consuming funds intended for national development.
Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids and flavonoids, which have been found to have antimicrobial properties in-vitro (Yang et al.,
2004). Onion bulbs contain a good number of phytochemicals, most of which are hydrocarbons and their derivatives (Griffiths et al., 2002) Several authors have reported pharmaceutical activity of extracts of A. cepa including anti-tumour, anti-diabetic, antioxidant, antimicrobial, anti-allergic and molluscicidal activity (Bakht et al., 2013); Griffiths et al., 2002). In vitro studies have shown that onion possess antibacterial, antiparasitic, and antifungal activity (Yang et al., 2004).
Herbs are cheap as compared to antibiotics and due to high antibiotic costs and bacterial resistance; people are opting for herbs in prevention and treatment. However, there is no standard dose on herbs hence the need for determination of concentration that can achieve a therapeutic effect. Therefore the project seeks to determine antibacterial properties of A. cepa against S. epidermidis and E. coli bacteria.
1.3: STATEMENT OF THE PROBLEM
Virulent bacterial strains are causative agents of various communicable diseases. Pathogenic
S. epidermidis bacteria cause most notably post-operative wound and soft tissue infections. E. coli causes mainly gastrointestinal tract infections. The thrust of the research is to determine antibacterial effects of raw A. cepa extract on S. epidermidis and E. coli bacteria.
1.4: JUSTIFICATION
The research seeks to determine antimicrobial properties of bulb onion thereby exploring benefits associated with regular onion use since it is cheap and easy to access. It is also
13 | P a g e envisaged that the research would help contribute to the body of knowledge on antimicrobial properties of A. cepa.
1.5 AIMS AND OBJECTIVES
1.5.1 AIM
To determine antibacterial activity of A. cepa extract on S. epidermidis and E. coli bacteria.
1.5.2 OBJECTIVES
1. To determine the antibacterial activity of fresh extract of A. cepa on S. epidermidis and E. coli bacteria.
2. To determine minimum inhibitory concentration (MIC) of A. cepa on S. epidermidis and E. coli bacteria.
3. To determine minimum bactericidal concentration (MBC) of A. cepa on S. epidermidis and
E. coli bacteria.
1.6 RESEARCH QUESTIONS
1. Does fresh A. cepa extract have antimicrobial effects on S. epidermidis and E. coli?
2. What is the minimum inhibitory concentration (MIC) of A. cepa on S. epidermidis and E. coli?
3. What is the minimum bactericidal concentration (MBC) of A. cepa on S. epidermidis and E. coli?
1.7: ASSUMPTIONS
The research assumed that;
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1. A. cepa has phytochemicals with antimicrobial effect on S. epidermidis and E. coli
bacteria.
2. Regular inclusion of raw A. cepa in diet and its external use helps in treatment and
prevention of S. epidermidis and E. coli bacterial infections.
1.8: LIMITATIONS
The research was done at the Bindura University of Science Education (BUSE) Astra laboratory, capable of handling only less infectious bacterial species. Suitable equipment like the antibiotic disk dispenser, antibiotic susceptibility disks were not available and hence improvisation was done. Recommended media for the tests: Mueller Hinton agar and broth was unavailable hence mannitol salt agar, nutrient agar and nutrient broth were used instead.
1.9: DELIMITATIONS
The research was restricted to two bacterial species, S. epidermidis and E. coli and only one onion species, A. cepa. The concerns of the research are only restricted to antimicrobial properties of A. cepa on S. epidermidis and E. coli
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CHAPTER 2: LITERATURE REVIEW
2.1 PLANT DESCRIPTION
Kingdom: Plantae
Phylum: Magnoliophyta
Class: Liliopsida
Order: Liliales
Family: Liliaceae
Genus: Allium
Species: A. cepa
The plant A. cepa, bulb onion, called hanyanisi and ithanga in Zimbabwean Shona and Ndebele languages is an annual vegetable plant used around the world. It has a fan of hollow, bluish green leaves. The bulb at the base of the plant begins to swell when a certain day length is reached. The bulb is utilised as food and it can be stored for prolonged periods after harvesting
(Block, 2010).
2.2 NUTRITIONAL CONTENT OF A. CEPA
The genus Allium consists of monocotyledonous flowering plants which include bulb onion (A. cepa), garlic (A. sativum), chives (A. schoenoprasum), shallot (A. ascalonium) and leeks (A. ampeloprasum) (Cutler and Wilson, 2004).
Plants of this genus produce chemical compounds, mostly derived from cysteine sulfoxides, which give them a characteristic alliaceous taste and odour (Block, 2010). The different types of alliums are used to spice up soups, stews, and salads. In many dishes both bulbs and leaves are used. Species of this genus have different taste when consumed. The difference is on ground sulphur content in which the species was cultivated (Cutler and Wilson, 2004).
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Alliums occupy a unique position both as edible food plants and herbal medicines (Burt, 2004).
Rose et al., (2005) assert that garlic is top selling herbal supplement of the genus and its products show considerable promise as environmentally friendly pesticides. The properties of
Alliums, specifically onions are based on the occurrence of a number of simple sulphur containing chemical compounds ingeniously packed by nature in these plants (Burt, 2004;
Griffiths et al., 2002). A substantial body of literature suggest that these sulphur containing compounds likely arose through natural selection for pest resistance (Yang et al., 2004).
Human consumption of Alliums therefore contributes to nourishment by providing these sulphur containing chemical compounds.
Sulphur obtained from the soil is used within plant cells to form amino acids: cysteine and methionine which in turn form the gamma glutamyl peptides (Yang et al., 2004). These peptides serve as building blocks for the allium flavour precursors like alliin (Block, 2010).
Alliin is present in mesophyll storage cells of each allium crop and determines its flavour.
When there is tissue disruption e. g by chopping, the enzyme allinase stored in the bundle sheath cells and protected from alliin by a membrane comes into contact and alliin is converted into allicin (Fossen et al., 1998). The volatile allicin causes an irritation of eyes when chopping onions (Salama et al., 2014). Thus the scent of an un-chopped onion bulb is completely different from the scent after tissues have been chopped, because of the enzymatic lysis
((Fossen et al., 1998).
According to, Salama et al., (2014) allicin exhibits antibacterial, antifungal, antiviral, antioxidant and antiprotozoal activity. The antimicrobial effect of allicin is due to chemical reaction with thiol groups of various bacterial enzymes e. g bacterial enzymes alcohol dehydrogenase, thioredoxin reductase and RNA polymerase (Block, 2010). Allicin is observed as Alliums defence mechanism against attack by pests (Yang et al., 2004).
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However, allicin is highly unstable and recently attention has been focused on the effects of phenolic compounds, such as flavonoids, which are more stable (Ioku et al., 2001). Bulb onion is known for being a natural source of flavonoids (Fossen et al., 1998). Flavonoids are known to have medicinal properties and hence consuming them can help in lowering various ailments.
Onions also contain fibre, vitamins C and B6 and are considered a low glycaemic food. The glycaemic index ranks carbohydrate containing foods based on how quickly they raise blood sugar levels. Moreover onions are free from, fat and cholesterol (Brouns et al., 2005).
2.3 MEDICINAL USES
The use of Alliums for medicinal purposes dates back at least 3500 years with mention of them in the ancient Egyptian Ebers Papyrus, a book of herbal knowledge, which documented their therapeutic use (Rivlin, 2001). Many researchers have tested the proposed medicinal attributes of Alliums and have validated them. According to Hedges and Lister (2007), including Alliums in diet is associated with: reduced risk of stomach cancer, brain cancer, reduced levels of cholesterol, triglycerides and thromboxane (substances involved in the development of cardiovascular disease) in blood. These medicinal properties have been attributed to flavonoids such as quercetin (Shon et al., 2004; Hirvonen et al., 2001; Kosmider and Osiecka, 2004).
Flavonoids are plant pigments which act as chemical messengers, physiological regulators and cell cycle inhibitors (Hirvonen et al., 2001). In onion bulbs, higher concentrations of quercetin occur in the outer most rings (Kosmider and Osiecka, 2004). Onion cannot be used as a substitute for therapy, but it may be of help to those who suffer from the mentioned ailments.
Onions have antioxidant activity when consumed which enables deactivation of free radicals and other oxidants, rendering them harmless (Shon et al., 2004). Free radicals which are
18 | P a g e obtained from either external sources like pollution, smoking or carcinogens in the environment can interfere with major life processes such as respiration (Hirvonen et al., 2001). Consuming onions can help in deactivating some of the free radicals from these external sources.
2.4 ANTIMICROBIAL PROPERTIES OF ALLIUM CEPA
2.4.1 ANTIBACTERIAL EFFECTS
In-vitro assays have shown that A. cepa has antimicrobial activity against some representative spoilage bacteria. Activity was attributed to the presence of organic-sulphur-containing compounds (Rivlin, 2001; Kim, 2009).
In another study by, Chun-Lin et al., (2012) A. cepa was found to have an essential oil with moderate antimicrobial activity against Escherichia coli, Bacillus subtilis and Streptococcus mutans. The MIC values ranged from 0.18 mg/mL to1.80 mg/mL and MBC from 0.54 mg/mL to 3.6 mg/mL on the three bacterial strains studied. Extracts from purple and yellow A. cepa extract were found to be bactericidal against clinical isolates of Vibrio cholerae. Purple type onion extract had MIC ranging 19.2–21.6 mg/ml and yellow type onion had an MIC ranging
66–68.4 mg/ml (Hannan et al., 2010). However, no MBC results were given in this study.
2.4.2 ANTIFUNGAL EFFECTS
Fresh onion bulbs in different solvents have been found to have in-vitro antifungal properties against Candida albicans (Bakht et al., 2013). These results agree with those reported by
Chaithradhyuthi et al., (2009) and Irkin and Korukllugin (2009).
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2.4.3 ANTIMICROBIAL PROPERTIES OF RAW AND COOKED ONION
In a study by Wilson and Demming-Adams, (2007), fresh onion was found to have antibacterial properties which were not found in cooked and stored onion.
While it is not possible to draw broad conclusions from a single lab study, these findings also suggested that length of storage (for raw chopped onion) and duration of exposure to heat (in this case involving exposure to steam for 10 full minutes) can affect some of onion's antibacterial properties (Kim, 2009; Wilson and Demming-Adams, 2007).
2. 5 TEST ORGANISMS USED IN THE RESEARCH
2.5.1 STAPHYLOCOCCUS EPIDERMIDIS DESCRIPTION
Kingdom: Eubacteria
Phylum: Firmicutes
Class: Coccus
Order: Bacillales
Family: Staphylococcaceae
Genus: Staphylococcus
Species: S. epidermidis
The discovery of the bacteria Staphylococcus epidermidis dates back to 1880 when Alexander
Ogston identified the major cause of pus in post-operative wounds (Newsom, 2008).
S. epidermidis is a gram positive, salt tolerant, non -motile and non-spore forming coccal bacteria. It is a facultative anaerobe which appears as grape like clusters when viewed under a microscope (Ryan and Ray, 2004). S. epidermidis reproduce asexually by binary fission. The
20 | P a g e two daughter cells do not fully separate but remain attached and hence they are observed as clusters (Kluytmans et al., 1997).
The bacterium is catalase positive and this has been used to distinguish it from enterococci and streptococci bacteria. Though it is part of the natural micro flora of humans, S epidermidis is responsible for many hospital acquired infections and skin problems like acne (Otto, 2009).
Tissue infection occurs when the skin and mucosal barriers have been breached.
Despite the availability of potent antimicrobial drugs, S epidermidis infection causes significant morbidity and mortality (Ryan and Ray, 2004). Virulence factors include the ability to form biofilms on inanimate objects probably because of the presence of surface proteins that binds blood and extracellular matrix proteins which allows other bacteria to bind creating a multilayer biofilm (Cenci-Goga et al., 2003).
Most S. epidermidis strains are slowly resistant to antibiotics such as penicillin and methicillin while much susceptibility is recorded to vancomycin. The methicillin resistant S. epidermidis is one of the greatly feared strains resistant to most B- lactam antibiotics (Waters et al., 2011).
Due to the rapid increase in resistance by S. epidermidis to conventional drugs, this project aimed at investigating the antibacterial activities of A. cepa on S. epidermidis so that people can maximise its use for treatment and prevention against infection.
2.5.2 ESCHERICHIA COLI DESCRIPTION
Kingdom: Eubacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
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Family: Enterobacteriaceae
Genus: Escherichia
Species E. coli
Escherichia coli are gram negative, facultatively anaerobic, rod shaped bacteria of the genus
Escherichia. The bacteria were discovered in 1885 by Theodore Escherich whilst on the hunt of the cause of fatal intestinal diseases in children (Donnenberg, 2013). Harmful strains of the species can be found lurking on foodstuffs producing toxins that result in food poisoning
(Todar, 2007). Symptoms of E. coli infection include abdominal pain, cramping, diarrhoea and vomiting (Hudault et al., 2001).
2.6 ANTIBACTERIAL AGENTS
2.6.1 CHARACTERISTICS OF ANTIBACTERIAL AGENTS
Antibacterial agents should have selective toxicity. This means that the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the patient. Clinically, useful antibiotics should have the following characteristics: wide spectrum of activity with the ability to destroy or inhibit many different bacterial species should not eliminate the normal flora of the host and should be able to reach part of the human body with infection (Rhee and
Gardiner, 2004). When being manufactured, the antibiotic should be inexpensive and easy to produce and should be chemically stable, i.e having a long shelf life (Mascio et al., 2007).
Proteins, nucleic acids and cell wall can be targets for antibiotics. Antibiotics such as penicillin, cephalosporin and vancomycin inhibit bacterial cell wall synthesis or activate enzymes that disrupt bacterial cell walls (Pankey and Sabath, 2004). Aminoglycosides, clindamycin and tetracycline inhibit protein synthesis or they induce the production of abnormal bacterial proteins. These bind irreversibly to bacterial ribosomes and when bound, the bacteria cannot
22 | P a g e synthesise proteins necessary for cell structures. Others disrupt microbial cell membrane and inhibit the organisms’ reproduction by interfering with nucleic acid synthesis (Mascio et al.,
2007).
2.6.2 MEDIA RECOMMENDED FOR SUSCEPTIBILITY TESTING
The recommended media for antimicrobial susceptibility testing is Muller-Hinton. Both agar and broth media are non-selective non-differential hence all organisms plated will grow and they have been thoroughly tested for their pH levels (between 7.2 and 7. 4). In addition, they contain starch which absorbs toxins released by cultured bacteria and therefore will not interfere with antibacterial agents. Lastly the agar allows for better diffusion of antimicrobials which leads to true zones of inhibition which can be reproduced from the same organism (Atlas,
2004).
2.6.3 METHODS USED TO DETERMINE SUSCEPTIBILITY OF BACTERIA TO
ANTIBACTERIAL AGENTS
In-vitro antimicrobial susceptibility testing should use validated methods. The goal of in-vitro antimicrobial susceptibility testing is to provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the infected host (Bonev et al., 2008). Available methods include the dilution method, disk diffusion method, automated antimicrobial susceptibility testing systems, mechanism- specific tests and genotypic methods (Bonev et al.,
2008). The selection of a method is based on many factors such as practicality, flexibility, automation, cost, reproducibility, accuracy, and individual preference (Jorgensen and
Turnidge, 2007).
Two methods that were used in this research are the Kirby- Bauer method (disk diffusion) and the tube dilution method.
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2.6.3.1 MCFARLAND TURBIDITY STANDARD
The McFarland turbidity standard allows one to work with a defined concentration of the test organism in order to get a confluent growth on the culture media plate (Hudzicki, 2009). The standards range from 0, 5 to 10 and the 0, 5 standard is recommended for the Staphylococcaceae and Enterobacteriaceae families (Jorgensen and Turnidge, 2007). The 0, 5 standard is equivalent to a bacterial density of approximately150 million colony forming units (CFU) per millilitre (Jorgensen and Turnidge, 2007).
2.6.3.2 KIRBY-BAUER METHOD
Kirby-Bauer is a qualitative method that looks at the diffusion of an antimicrobial agent of a specified concentration from disks, tablets or strips, into the solid culture medium that has been seeded with the selected inoculum isolated in a pure culture (Bonev et al., 2008).
All aspects of the Kirby- Bauer method are standardised to ensure consistent and accurate results. Only young pure cultures with inoculum size standardised against the McFarland’s turbidity standard are used. Zones of inhibition are influenced by several factors and these include the concentration of bacteria on spread plate, susceptibility of the bacteria to the antibacterial agent, drug or herb antagonists, nutrient availability, agar depths and growth temperature (Hudzicki, 2009). The antibacterial agent diffuses into the media inoculated with the test microorganism and its diffusion is influenced by its concentration, solubility in water and molecular weight (Bonev et al., 2008). Large molecules will diffuse at a slower rate than lower molecular weight compounds (Hudzicki, 2009).
The size of zone of inhibition is used to determine whether bacteria are sensitive or resistant to the antibacterial agent (Wiegand et al., 2008). Table1 below shows the zone interpretive chart adapted from (Hudzicki, 2009).
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Table 1: Zone size interpretive chart
Zone size produced (mm)
Bacteria Disc content Resistant Intermediate Susceptible
S. epidermidis 10 units ≤ 20 21-28 >29
E. coli 10 units ≤ 11 12-21 >22
2.6.3.3 TUBE DILUTION METHOD
The bactericidal and bacteriostatic effect of the antimicrobial is determined using the tube dilution method (Aneja, 2005). This is a quantitative method used to determine the minimum effective concentrations that is the MIC and MBC. MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism
(Andrews, 2001). MIC is the concentration of antimicrobial required to halt microbial growth for as long as it is present but if removed, growth resumes (Aneja, 2005). The MIC is correlated with the concentration of the antibiotic achievable in blood. Clinically, MIC is used not only to determine the amount of antibiotic that the patient will receive but also the type of antibiotic used, which in turn lowers the opportunity for microbial resistance to specific antimicrobial agents (Davison et al., 2000).
MBC is determined from broth dilution MIC tests by sub-culturing to agar plates that do not contain the test agent. MBC is the lowest concentration of an antimicrobial agent required to kill particular bacteria (Davison et al., 2000). The tube dilution method however requires strictly aseptic conditions as any chance of contamination may completely invalidate results.
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CHAPTER 3: RESEARCH METHODOLOGY
3.1 STUDY SITE AND SAMPLE COLLECTION
Fresh A. cepa bulbs were bought from Bindura vegetable market for lab use. A pure culture of
S. epidermidis was obtained from Bindura Hospital laboratory and E. coli from Bindura
University of Science Education Astra laboratory in Mashonaland West province of
Zimbabwe. All laboratory work was done at the same laboratory. Though the test organisms require clinical labs, they can be worked on in well-equipped labs since they are not highly contagious.
3.2 PREPARATION OF ALLIUM CEPA EXTRACT
The A. cepa bulbs were rinsed thoroughly in sterile distilled water to remove any microbes and dirty on surface. Microbes on the surface would contaminate the extract producing toxins or antimicrobials which could invalidate results. The outer covering and roots of the bulbs were manually peeled off and the fleshy part of the onion re-washed, sliced and then air dried.
70g of the onion bulb were aseptically crushed using sterile motor and pistil. The resultant paste was dissolved in 200ml sterile distilled water, giving a concentration of 350mg/ml. The mixture was allowed to stand for 1 hour and then filtered. It was noted during lab work pre- tests that A. cepa extract changes colour when stored overnight indicating a very short shelf life, hence new bulbs from same source were processed on each trial.
3.3 PREPARATION OF MEDIA AND SOLUTIUONS
3.3.1 MANNITOL SALT AGAR
One hundred and twenty grams of mannitol salt agar powder was suspended in a litre of distilled water. The mixture was boiled while stirring until completely dissolved. The media was sterilised by autoclaving at 121°C for 15 minutes. It was cooled to 44, 5°C in a water bath
26 | P a g e and then aseptically poured into sterile petri dishes. The plates were sealed with parafilm and stored at 4°C in the dark and used within 7 days of preparation.
3.3.2 NUTRIENT AGAR
Twenty eight grams of nutrient agar was suspended in a litre of sterile distilled water, stirred and boiled to dissolve. The media was sterilised by autoclaving at 121°C for 15 minutes. It was cooled to 50°C in water bath and then aseptically poured into sterile petri dishes. The plates were sealed with parafilm and stored at 4°C in the dark and used within 3 days of preparation to avoid contamination associated with lengthy storage of this general purpose media
(MacFaddin, 2000).
3.3.3 NUTRIENT BROTH
A total of 21g nutrient broth was suspended in a litre of sterile distilled water and swirled until completely mixed. This was then dispensed in 9ml volumes into clean capped tubes using a sterile syringe. The tubes were autoclaved at 121°C for 15min. The broth was kept aseptically in these capped tubes at 4°C in the dark and used within 3 days.
3.3.4 PREPARATION OF PENICILLIN G SODIUM SALT SOLUTION
A total of 6.4 g of penicillin G sodium salt was dissolved in 100ml sterile distilled water to give a concentration of 64 mg/ml.
3.4 PREPARATION AND MAINTANANCE OF TEST ORGANISMS
3.4.1 STAPHYLOCOCCUS EPIDERMIDIS
The S. epidermidis sample was aseptically sub-cultured by streaking on sterile mannitol salt agar plates and incubated at 37°C for 24 hours in a NUVE EN 500 type incubator. Sub- culturing was done to obtain a pure culture. Pure cultures were stored in the refrigerator at 2°C
27 | P a g e for further use. On each trial the stored sample was sub-cultured onto sterile mannitol salt agar plates.
3.4.2 ESCHERICHIA COLI
The E. coli sample was aseptically sub cultured by streaking on nutrient agar several times until pure cultures were observed. The pure cultures were stored in the refrigerator at 2°C for further use. On each trial the stored sample was sub-cultured onto sterile nutrient agar plates.
3.5 IDENTIFICATION OF CULTURES USED AS TEST ORGANISM
3.5.1 STAPHYLOCOCCUS EPIDERMIDIS
3.5.1.1 GROWTH ON MANNITOL SALT AGAR
S. epidermidis was grown on mannitol salt agar. Mannitol salt agar is a selective and differential media inhibiting growth of most bacterial mutants and selective to staphylococci species like
S. epidermidis because of its high sodium chloride concentrations (Anderson, 2013). From the pure sub-cultured colonies obtained, a gram stain reaction was used to morphologically identify the test organism.
3.5.1.2 GRAM STAIN
Gram stain method differentiates bacteria by chemical and physical properties of their cell walls. It detects peptidoglycan, which is present as a thick layer in gram positive bacteria and thin in gram negative bacteria. A 24 hour plate culture of test organism was used for the test.
A sterile, dry, grease or oil free slide was labelled. Using a sterile cooled loop, a drop of distilled water and two colonies were aseptically transferred on to the slide. A smear was made and allowed to air dry. Smears typically require only a small amount of bacterial culture; as a thick
28 | P a g e smear diminishes the amount of light that can pass through, thus making it difficult to visualise the morphology of single cells (Beveridge, 2001).
The smear was heat fixed on a Bunsen flame. Heat fixation kills the bacteria on the smear, enables adherence of smear to the slide and allows sample to more readily take up stains (Black,
2012). The slide with a heat fixed smear was flooded with crystal violet and allowed to stand for 1 minute. The slide was rinsed with distilled water. The slide was flooded with Grams iodine and left to stand for 2 minutes. Iodine acts as a mordant and helps fix the dye by combining with the crystal violet in cells forming crystal violet iodine complex compound. The slide was tilted and rinsed with distilled water. The smear appeared as a purple circle on the slide.
Using 95% ethyl alcohol, the smear was decolourised after which rinsing with distilled water was done. Counterstaining with a few drops of safranin was done and slide left to stand for 40 seconds and washed with distilled water. A paper was used to blot dry the slide and finally viewing under the light microscope at high magnification with oil immersion was done. The reagents used in gram staining are given in the appendix.
3.5.2 ESCHERICHIA COLI
3. 5.2.1 GROWTH ON NUTRIENT AGAR
Observations of E. coli growth on nutrient agar were used as the basis to confirm this test organism. Nutrient agar is a general purpose growth medium and hence thorough visualisation of creamy white colonies would confirm uncontaminated E. coli identity. A gram stain reaction was used to morphologically identify the test organism.
3.5.2.2 GRAM STAINING
The gram stain was done as mentioned in section 3.5.1.2.
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3.6 PREPARATION AND MAINTENANCE OF STOCK CULTURE
3.6.1 STAPHYLOCOCCUS EPIDERMIDIS
Microbial stock culture was prepared by aseptically inoculating the test microorganism, S. epidermidis into prepared mannitol salt agar plates to maintain a pure culture since the media is selective. The sterile streaked plates were then sealed with parafilm to avoid contamination and also to slow drying. The plates were incubated for 24 hours at 37°C and then stored at 2°C in a refrigerator. The cultures were used within a week of preparation. Stock cultures were done solely to keep the organism viable for subculture into fresh medium.
3.6.2 ESCHERICHIA COLI
The bacteria, E. coli stock culture was prepared by aseptically inoculating it into sterile nutrient agar plates, sealing with parafilm, incubating at 37°C for 24 hours and then storing plates in refrigerator at 2°C.
3.7 ANTIBACTERIAL TESTS
The Kirby-Bauer tests were done and their purpose was to determine the antibacterial properties of A. cepa extract on S. epidermidis and E. coli. In both tests, the experiments were concurrently run with two controls: the positive and negative control. The only available antibiotic in the Biological Sciences laboratory was penicillin G sodium salt and this was used as the positive control. Penicillin G sodium salt has recorded sensitivities against S. epidermidis and E. coli though resistance has been reported (Joseph and Kosinski, 1996). Sterile distilled water was used in the negative control, to establish whether aseptic techniques had been maintained in the experiment. Thus, it was a sterility control.
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3.7.1 MCFARLAND TURBIDITY STANDARD PREPARATION
The 0.5 McFarland Equivalence Turbidity Standard was prepared and used to adjust densities of bacterial suspensions used for identification and susceptibility testing (Wiegand et al., 2008).
Half a millilitre of 1 % w/v barium chloride solution was added to 99.5ml of 1% v/v sulphuric acid to give a barium sulphate precipitate which is the 0.5 turbidity standard precipitate. This standard should appear turbid with a white suspension upon agitation and should have an absorbance of between 0.08 and 0.1 at 625nm (Pankey and Sabath, 2004). The absorbance of the prepared McFarland turbidity standard solution was measured using a Genesys 10s UV-
Vis spectrophotometer. The standard was stored in the dark at 20°C.
3.7.2 USE OF 0.5 MACFARLAND STANDARDS
Isolated colonies of the S. epidermidis and E. coli were separately added to sterile uninnoculated nutrient broth and incubated at 37°C for 24 hours to obtain a moderate turbidity.
The inoculum was agitated after incubation. Visual comparison of the turbidity of the tubes was done under adequate light with tubes being read against a white card with contrasting black lines (Wickerham card). Equal obliteration or distortion of black lines indicated a turbidity match between bacterial suspension and the McFarland standard. The approximate cell density equivalent to the 0.5 McFarland standards is 150 million colony forming units (CFU) per millilitre (Pankey and Sabath, 2004). The standardised cultures were used in all tests.
3.7.3 DISK DIFFUSION TEST
3.7.3.1 PREPARATION OF DISKS
20 mm diameter filter paper disks were prepared by cutting using scapel blade. These were wrapped in aluminium foil paper and sterilised in a dry heat oven at 105°C for 1 hour.
3.7.3.2 SUSCEPTIBILITY TEST FOR S. EPIDERMIDIS
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Mannitol salt agar plates prepared in 3.3.1 were aseptically allowed to come to room temperature. They were then inverted to allow excess liquid drain from the agar surface. A total of 10 plates were labelled. A sterile cotton swab was dipped into the standardised S. epidermidis inoculum tube. The swab was dragged against the side of the tube (above fluid level) using firm pressure to remove excess fluid. The swab was rubbed over the entire surface of agar plate, rotating the plate each time to ensure an even distribution of the inoculum. The swab was discarded into an appropriate container after use.
The plates were left at room temperature for 10 min for the surface of the agar plate to dry and facilitate absorption of inoculum into agar (Bonev et al., 2008).
Sterile 20mm filter paper disks were aseptically dispensed directly onto the inoculated agar using sterile forceps. A filter paper disk was placed at the centre of each plate. Using a sterile pipette, 1.0ml of A. cepa extract was dropped on each disk giving a disk concentration of
1.75mg/ml. Each disk was gently tapped with sterile forceps to ensure contact with agar surface. The plates were incubated at 37°C for 24hours (Bonev et al., 2008).
One millilitre of penicillin G sodium salt was dropped on the filter paper disk on agar spread with inoculum; giving a disk concentration of 0.64mg/ml. Steps similar to those of A. cepa disks were followed. The negative control plates had no inoculum and filter paper disks were impregnated with sterile distilled water and the plates were incubated.
3.7.3.3 SUSCEPTIBILITY TESTS FOR E.COLI
Testing for the susceptibility of E. coli to A. cepa was done using nutrient agar and procedures followed were similar to those done on S. epidermidis stated in 3.7.3.2 above.
3.7.4 TUBE DILUTION TESTS
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Tube dilution tests were done to determine the MIC and MBC. Due to limited resources tube dilution tests were only done for S. epidermidis
3.7.4.1 DETERMINATION OF MIC
Sterile capped tubes with nutrient broth were numbered 1-10. Serial dilution was done as follows: Using a sterile pipette and maintaining aseptic techniques, 1.0ml A. cepa extract of concentration 1.75mg/ml was added to tube 1 and well mixed. The pipette was discarded and using another pipette, 1.0ml from the first test tube was transferred to the second tube. Contents of second tube were mixed and using a separate pipette, 1.0ml was transferred to the third tube.
Dilutions were continued in this manner up to tube 9, changing pipettes between tubes. 1.0ml from tube 9 was removed and discarded. The tenth tube was left as the growth control and did not receive A. cepa extract. Using a new sterile pipette, 1.0ml of inoculum standardised against
0.5 McFarland standards was added to each of the tubes aseptically.
Using other sterile tubes and maintaining aseptic techniques, the process was repeated using the antibiotic penicillin G sodium salt as the positive control and using sterile distilled water as the negative control. All the tubes were incubated at 37º C for 24 hours and then examined for bacterial growth to determine the MIC. Results were recorded as (+) where there was turbid growth and (-) where there was no any detectable growth. The last bottle showing no growth was taken as the MIC and results of the MIC would be valid only if the control tubes had no growth (Jorgensen and Turnidge 2007).
3.7.4.2 DETERMINATION OF MBC
From each tube that showed no growth in the MIC tests, a loopful of culture was aseptically removed and streaked onto nutrient agar. There was change of media from mannitol salt agar to nutrient agar because the earlier was used up. The plates were divided into quadrants and
33 | P a g e the loopful from each tube was streaked onto the different quadrants. The different quadrants were labelled according to the concentrations in the tube from which inoculum was taken. The plates were sealed, inverted and incubated at 37°C and then checked for growth after 24 hours.
The last quadrants showing no growth were taken as the minimum bactericidal concentration
(Cushnie and Lamb, 2011).
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CHAPTER 4: RESULTS
4.1 IDENTIFICATION OF BACTERIAL CULTURE
4.1.1 STAPHYLOCOCCUS EPIDERMIDIS
In mannitol salt agar, S. epidermidis grew producing small pink to red colonies with no colour change to the medium. This showed the culture was S. epidermidis, species that do not ferment mannitol hence no colour change to the medium. Gram stain results showed the test organism was gram positive. Clusters of cells in a deep violet colour were observed. Formation of clusters indicated the bacterial sample was of the genus Staphylococcus.
4.1.2 ESCHERICHIA COLI
The bacteria grew on nutrient agar showing creamy white colonies. Gram stain results showed distinct rods in red colour.
4.2 0.5 MCFARLANDS TURBIDITY STANDARD
The absorbance of the prepared 0.5 McFarland Equivalence turbidity standard was measured and fig 1 below shows the results with tested absorbance of 0.1.
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Figure 1: Absorbance values for McFarland standard.
4.2.1 DISK DIFFUSION TEST
4.2.1.1 S. EPIDERMIDIS
S. epidermidis produced no inhibition zone sizes with A. cepa (fig 2) but with penicillin G sodium salt, S. epidermidis produced inhibition zones (fig 3). There was no growth in the sterility, negative control plates.
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Fig 2: Activity of A. cepa against S. epidermidis
Fig 3: Inhibition zones of penicillin G sodium salt against S. epidermidis.
The diameters of the zones of inhibition formed by A. cepa and penicillin G sodium salt against
S. epidermidis were recorded in table 2 below.
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Table 2: Inhibition zone diameters of S. epidermidis with A. cepa extract and penicillin G sodium salt.
Zone diameters produced (mm)
Test agent Plate 1 Plate 2 Plate 3 Mean Standard deviation
A. cepa 0 0 0 0 0
Penicillin G sodium salt 22 21 22 21.66 ±0.577
4.2.1.2 E. COLI
E. coli was found to be susceptible to both A. cepa and penicillin G sodium salt as shown in figures 3 and 4. Sterility control plates showed no growth.
Fig 4: Inhibition zone of A. cepa against E. coli.
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Fig 5: Inhibition zone of penicillin G sodium salt against E. coli.
The diameters of zones of inhibition formed by A. cepa and penicillin G sodium salt against E. coli were recorded in table 3 below.
Table 3: Inhibition zone diameters of E. coli with A. cepa and penicillin G
sodium salt
Test agent Zone diameter produced (mm)
Trial 1 Trial 2
Plate 1 Plate 2 Plate1 Plate2 Mean Standard deviation
A. cepa 20 23 33 30 26.5 ± 6.02
Penicillin G 31 29 34 33 31.75 ± 2.21
sodium salt
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4.2.2 TUBE DILUTION TESTS
Tube dilution tests were done only for the species S. epidermidis due to limited resources. Raw data on MIC of A. cepa and penicillin G sodium salt are shown in appendix 3. The MIC and
MBC of A. cepa and penicillin G sodium salt on S. epidermidis were recorded in tables below.
No growth was observed in growth control tubes.
Table 4: Mean MIC and standard deviations of A. cepa and penicillin G sodium salt on
S. epidermidis.
Test agent Concentration of agent (mg/ml)
Trial 1 Trial 2 Mean Standard deviation
A. cepa 0.0175 0.175 0.096 ±0.11
Penicillin G sodium salt 0.0064 0.0064 0.0064 0
Table 5: Mean MBC and standard deviations of A. cepa and penicillin G sodium salt on
S. epidermidis
Test agent Concentration of agent (mg/ml)
Trial 1 Trial 2 Mean Standard deviation
A. cepa 1.75 1.75 1.75 0
Penicillin G sodium salt 0.0064 0.064 0.035 ±0.04
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CHAPTER 5: DISCUSSION AND CONCLUSION
5.1 DISCUSSION
The MIC values obtained were valid since no growth was observed in the sterility control tubes and standardised pure inocula of E. coli and S. epidermidis were used. The inocula were standardised using the 0.5 McFarland turbidity standards. The standard should have an absorbance of approximately 0.1 at 600-625nm (Pankey and Sabath, 2004). Sterility control tubes were used to determine if contaminants were encountered during the tests.
Penicillin G sodium salt, an antibiotic to which S. epidermidis and E. coli are slowly resistant to was used. This was the only available antibiotic. Methods used for antibacterial susceptibility tests include the dilution method, disk diffusion method, automated antimicrobial susceptibility testing systems, mechanism- specific tests and genotypic methods. The disk diffusion and dilution methods were found suitable for the research because they were flexible and cost effective (Jorgensen and Turnidge, 2007).
There were no inhibition zones formed by A. cepa against S. epidermidis but using the tube dilution method, an MIC (mean 0.096, SD ±0.11) was obtained. Mannitol salt agar used in disk diffusion and nutrient broth used in serial dilutions have considerable differences in pH and nutrient availability ( Andrews, 2001).Agar and broth media have different states hence diffusion of antimicrobials will be different. Interaction between media and test agent differ and molecules in broth media diffuse faster than those in agar media. The results differ from those by Hannan et al., (2010) which showed that purple type bulb onion extracts had antibacterial activity against V. cholerae, mean 25.83; SD ±2.18 at 100% concentration.
In addition, media recommended for disk diffusion tests, Mueller Hinton agar has the following characteristics not found in mannitol salt and nutrient agar used in the research. It is non- selective and not differential, contains starch which absorbs toxins released by cultured bacteria
41 | P a g e and allows for better diffusion of antimicrobials. These lead to true zones of inhibition which can be reproduced from the same organism (Atlas, 2004).
Showing no inhibition zones could also be due to molecular size of agent and media. Agents having large molecules with large molecular weights diffuse slowly through agar media than those with small molecules (Wiegand et al., 2008). Zone sizes are also influenced by agar depth since the antimicrobial diffuses in three dimensions, thus a shallow layer of agar will produce a large zone of inhibition than a deeper layer (Wiegand et al., 2008). Tests therefore require agar of the same depth.
A. cepa had higher MIC and MBC values than penicillin G sodium salt against S. epidermidis.
This shows A. cepa is a weak antimicrobial and could be because the extract has many components unlike penicillin G sodium salt with known concentration of the active antimicrobial component. Penicillin G sodium salt has known capabilities to interfere with bacterial cells thus causing cell death (Pankey and Sabath, 2004). Herbs have no known definite mode of action on microorganisms. This therefore could have contributed to large inhibition zone sizes of penicillin G sodium salt than of A. cepa on E. coli.
Inhibition zones formed by penicillin G sodium salt (31.75; SD ± 2.21) against E. coli are larger than those formed by A. cepa (26.5; SD ± 6.02). These results are comparable to those of Chun-
Lin et al., (2012). Bulb A. cepa oil had antimicrobial activities against Escherichia coli,
Bacillus subtilis and Streptococcus mutans. Bulb extracts are more effective as antibacterial agents as they have higher concentrations of allicin than other organs (Chehregani et al., 2007
Penicillin G sodium salt had small inhibition zone sizes on S. epidermidis (mean 21.66, SD
±0.577) than those of E. coli (31.75, SD ± 2.21). This could be due to the nature of S.
42 | P a g e epidermidis forming biofilms that cause delayed penetration of the antimicrobial into biofilm extracellular matrix (Stewart, 2002). Moreover this could be as a result of the differences in media used. Mannitol salt agar is a selective and differential media while nutrient agar is a general purpose media hence their composition can have an effect on diffusion of antibacterial agents. The use of the media is however justified.
The difference in mean MIC values shows the need for determination of concentration of herbs that can achieve a therapeutic effect. In the study, white bulb onion was used and differences in findings to those of Hannan et al., (2010) could be an indication of phytochemical differences among types of A. cepa species.
The activity of A. cepa could have been affected by solvent used in extraction of plant material.
A water extracted sample was used while literature shows use of solvents like petroleum ether, ethyl acetate, chloroform and butanol (Bakht et al., 2013). Conclusions cannot be drawn from a single lab study. However water extracted samples of A. cepa had no effect on the growth of
Candida albicans at any concentration (Bakht et al., 2013).
Plant extracts have merits over conventional drugs. Plant extracts are readily available and are easy to prepare. Over and above that, plant extracts have no recorded cases of overdose, overuse and side effects. However there is need to check if herbs like A. cepa have antagonistic or synergistic effects.
As less data is available regarding antimicrobial effect of A. cepa and other herbs against S. epidermidis, effects against E. coli have been determined in various studies. It is hoped that the study would contribute and lead to establishment of more potent antimicrobial substances of natural origin.
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5.2 CONCLUSION
S. epidermidis and E. coli infections are a cause for concern in Zimbabwe and globally. Due to increase in bacterial resistance and the inability to access antibiotics, natural, herbal plants can be used in prevention and treatment. Herbal plants like A. cepa extract with antimicrobial effects against S. epidermidis and E. coli can be used. There is need to determine a concentration of A. cepa extract that can achieve a therapeutic effect.
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APPENDICES
APPENDIX 1
Preparation of chemicals used for McFarland Equivalence Turbidity standard
1. 1% w/v Barium chloride solution
0.5g dihydrate barium chloride salt dissolved in 50ml distilled water.
2. 1% v/v Sulphuric acid solution
1ml concentrated sulphuric acid was dissolved in 99ml distilled water
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APPENDIX 2
List of reagents for Gram staining
Primary stain- crystal violet
Mordant- Grams iodine
Decolouriser- ethyl alcohol
Secondary stain- safranin
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APPENDIX 3
a) The MIC of A. cepa extract against S. epidermidis
Con (mg/ml) (n)10-1 (n)10-2 (n)10-3 (n)10-4 (n)10-5 (n)10-6 (n)10-7 (n)10-8 (n)10-9 (n)10-10
Trial 1 - - + + + + + + + +
Trial 2 - + + + + + + + + +
Negative ------
Control
Key: (n) = 1.75mg/ml
- = Not turbid
+ = Turbid b) The MIC of penicillin G sodium salt against S. epidermidis
Con (mg/ml) (n)10-1 (n)10-2 (n)10-3 (n)10-4 (n)10-5 (n)10-6 (n)10-7 (n)10-8 (n)10-9 (n)10-10
Trial 1 - - + + + + + + + +
Trial 2 - - + + + + + + + +
Negative ------
Control
Key: (n) = 0.64mg/ml
- = Not turbid
+ =Turbid
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