ANTIBACTERIAL ACTIVITY OF CLOVES (Syzygium aromaticum)AGAINST Staphylococcus aureus AND Escherichia coli

ANTIBACTERIAL ACTIVITY OF CLOVES (Syzygium aromaticum)AGAINST Staphylococcus aureus AND Escherichia coli

                               AGAINST   Staphylococcus aureus AND Escherichia


ADM. NO: 1510204024

I dedicate this work to Almighty ALLAH for giving me the ability and mental strength to have reached my final year in my academic career and also to my humbled parent, brothers, sisters and friend for their huge assistance and support financially. May ALLAH bless them all (Ameen).

In the name of Allah the most beneficial the most merciful. All praise is for Allah (SWT), and may the peace and blessing of Allah be upon his prophet Muhammad (SWA) and his companions and all those who follow them and their way until day of resurrection Ameen.
I sincerely express my profound gratitude to my supervisor DR A.A FARUK for his helpful suggestion and excellent supervisory role, May Allah continues to guide him through his work Ameen.
My deepest appreciation goes to my lovely and caring parents(Alh.Umar Muhammad Dakingari and Haj.Aisha Usman Dakingari) thank you for being there for me always, may Allah protect, guide and reward them with jannatul firdaus Ameen.
Also my respect goes to my brothers (Abba, Suleiman, and Usman) sisters(Ummi, Najaat and Nana khadijat) and friends Abber G, Farak, zamau, wale and my lovely uncle (Ibrahim) and aunt (aunty kulu) thank you all for your financial support.
My special thanks goes to all my lecturers for their hard work, dedication and contribution to my studies thank you all and may Allah protect their image upwards.

Table of Contents
1.1 Statement of Research Problem 3
1.2 Justification 3
1.3 Aim 3
2.1 Scientific Classification of Syzygium aromaticum 5
2.2 Description of cloves 5
2.3 Climate and soil 6
2.4 Nutrient Content of Cloves 6
2.5 Chemical Constituents 7
2.5.1   Bud Oil 8
2.5.2     Non-volatile 8
2.5.3 Tannins 9
2.5.4 Flavonoids 9
2.6 Medical Uses 9
2.7      Traditional Medicinal Uses 11
2.8 Description Of Staphylococcus Aureus 11
2.8.1 Scientific Classification Of Staphylococcus Aureus 13
2.9 Description of Escherichia Coli 13
2.9.1 Scientific Classification 0f Escherichia Coli 14
3. 1 Sample Collection 15
3.2 Preparation of the Plant Extract 15
3.3 Collection of Test Organism 15
3.4 Qualitative Phytochemical Analysis 16
3.4.1 Test for Tannins 16
3.4.2 Test for Flavonoids 16
3.4.3 Test for Saponins 16
3.4.4 Test for Glycosides 16
3.4.5 Test for Phenol 16
3.4.6 Test for Terpenoids 17
3.5 Preparation of Mueller Hilton agar 17
3.6 Preparation of nutrient agar 17
3.7 Sensitivity Test Using Agar well diffusion method 17
3.8   Minimum Inhibitory Concentrations and Minimum Bacteriocidal Concentration 18
4.0 RESULTS 19
4.1 Phytochemical properties of plant extract of cloves(Syzygium aromaticum) 19
4.2 Antibacterial Activity 19
Table 4.2 Clove sensivity result of   Staphylococcus aureus 21
Table 4.2.1 Clove sensitivity result of  Escherichia coli 22
Table 4.3 Shows growth on test tube (turbidity) and ager plate 23
5.1 Conclusion 25
5.2 Recommendation 25


This research was aimed at determining the antibacterial activity of clove against Staphylococcus
aureus and Escherichia coli. Clove was obtained in its dried form Central Market, Birnin kebbi. The test bacteria was obtained from Kebbi State University of Science and Technology Aliero. Agar well diffusion method of sensitivity test was adopted. From the result, The Zone of inhibition of Cloves against Staphylococcus aureus was found to be 20mm, 16mm, 15mm, 5mm, 4mm and 2mm by  .While on Escherichia coli ,Clove had zone of inhibition of  were  19mm, 18mm, 16mm, 5mm, 3mm and 2mm. The minimum inhibitory concentration (MIC) was 20mg/ml, and the Minimum bacteriocidal concentration was lOmg/ml. The Phytochemical components determined in Cloves were Saponins, Phenols, Tannins, Flavonoids, GIycosides and Terpeniods. Therefore

                                                       CHAPTER ONE
  Plants supply peoples with food, clothing and shelter. Many of our most useful medicines are also made from plants. In addition, plants add beauty and pleasure to our lives; most people enjoy the smell of flowers. The sight of a field of waving grain and quiet within a forest (Sharma et al., 1993).Plants remain the most common source of antimicrobial agents. Their usage as traditional health remedies is the most popular for 80% of world population in Africa, Asia and Latin America and is reported to have minimal side effect (Bibitha et al., 2002).
Many useful drugs come from plants. Some of these plants have been used as medicines for hundreds of years. More than 400 years ago, for example, some Indian tribes of South America used the bark of cinchona tree to reduce fever, the bark is still used to make quinine, a drug used to treat malaria and other disease. Chemical compounds in plants mediate their effect on human body through processes identical to those already well understood for the chemical. Compounds in conventional drugs, thus herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be effective as conventional medicines, but also gives them the same potential to cause harmful side effect (Lai and Roy, 2004). Man has use plant to treat common infectious disease and some of various maladies (Jung et al, 1991)
Cloves (Syzygium aromaticum) are the aromatic flower buds of a tree in the Family myrtaceae, they are native to the Maluku islands, and are commonly used as a spice. Cloves are commercially harvested primarily in Indonesia, India, Madagascar, Zanzibar, Pakistan Sri Lanka and Tanzania (Bullock and Harrison, 2005). Spices as cloves, ore medicinal, thyme and cinnamon, have been employed for centuries as food preservatives and antibacterial, plants mainly due to its antioxidant and antimicrobial activities. Nowadays, many reports confirm the antibacterial, antifungal, antiviral and anti-carcinogenic properties of spice plants. Cloves in particular have an antimicrobial attracted the attention due to the potent antioxidant activities standing out among the other spices (Shan et al, 2005).  Cloves are available throughout the year due to different
harvest seasons in different countries. (Yun and Wanjung, 2013). The clove tree is an evergreen that grows up to 8–12 m tall, with large leaves and crimson flowers grouped in terminal clusters. The flower buds initially have a pale hue, gradually turn green, then transition to a bright red when ready for harvest.
   Spices have been defined as plant substances from indigenous or exotic origin, aromatic or with strong taste, used to enhance the taste of foods (Arora and Kaur, 1999)(Shelef 1998).The active ingredients of plants against microorganisms are mostly some of the secondary metabolites (i.e. alkaloids, glycosides etc.) that are present in abundance in herbs and spices commonly used in Indian food preparations. Traditional medicines have been used for many centuries by a substantial proportion of the population of India [].Syzygium species (Fam. Myrtaceae) have been reported to possess antibacterial and anti- inflammatory activity (Muruganada et al., 2001). It was reported that the buds of Syzygium aromaticum (L.) Merr. & Perry (clove) were used in folk medicine as diuretic, odontalgic, stomachic, tonicardiac, aromatic condiment properties and condiment with carminative and stimulant activity(Boulos 2000). Syzygium aromaticum, commonly called cloves, and locally called ‘kenepeli’ and ‘kanumfari’ by the Igala and Hausa ethnic groups of central and Northern Nigeria, respectively, is a common spice used in local beverages in the middle belt and northern part of the country (Atawodi et al., 2011).The clove tree is an evergreen tropical plant, which flowers twice every year. Cloves are the unopened buds and harvested when the outer green leaves (calyx) have changed from green to a yellow pink(Ahmad and Beg, 2001).

1.1 Statement of Research Problem
Infectious diseases are world leading cause of premature death, killing almost 50,000 peoples every day. Resistance to antimicrobial agent is emerging in a wide variety of pathogens and multiple drug resistance (MDR) is becoming common in diverse organisms. This is a cause for concern as many microorganisms hitherto susceptible to antimicrobial agent are becoming resistant with devastating consequences on health and well being of individuals.
1.2 Justification
A clove (Syzygium aromaticum) contains some constituents which have bactericidal effects on a wide range of organisms, so this experiment is carried out to exploit this natural phenomenon to create a new antibiotic.
1.3 Aim

The aim of the research is to determine   the antibacterial activity of   Cloves on Staphylococcus  aureus  and Escherichia coli.

The objectives are to:
 1.   Carry out phytochemical screening of cloves.
  2.  Determine the antibacterial activity of Crude extract of Clove against Staphylococcus aureus  and Escherichia coli.
  Determine   the   Minimum   inhibitory   concentrations(MIC)   and   Minimum bactericidal concentrations(MBC)

                                     CHAPTER TWO
    Traditional therapy involves the use of plants extracts or their active principles which may serve as a source of modern drugs source and source of intermediate compounds for synthesizing analog drugs with more desirable properties (Akerele, 1998). The development of novel and more active therapeutic agents, further investigation in the chemical and biological activities of the plant needed to be carried out (Roja and Rao, 2002). The work is to evaluate the antibacterial activity of cloves extract against staphylococcus aureus and Escherichia coli dried flower buds, leaves and stems to make medicine. Clove is used as an expectorant. Expectorant make it easier to cough up phlegm; clove oil is used for diarrhea, hernia and bad breath. Clove and clove oil are used for intestinal gas, nausea and vomiting. It is also applied to the skin as a contenritant for pain and for mouth and throat inflammation (Irene, 2013). In manufacturing, clove is use in toothpaste, soap, cosmetics, perfumes, and cigarettes. Clove cigarettes, also called kreteks, generally contains 60% to 80% tobacco and 20% to 40% ground clove. Eugenol one of the chemicals in clove acts like menthol to reduce the harshness of tobacco smoke (Merry et al,. 2011). Clove is an evergreen tropical tree with strong antioxidant activity and also reported have the strongly antioxidant activity among spices tested. Clove have been reported to have antifungal, antiviral, anti-inflammation and antioxidant properties (Pohlit et al, 2011).
The clove is an evergreen grows up to 8- 12m tall, with tree that large leaves and crimson flowers grouped in terminal clusters. The flower buds initially have a pale hue,, gradually turn green, and then transmitted to bright red when ready for harvest. Cloves are harvested at 1.5-2.0cm long, and consist of a long calyx that terminates in four spreading sepals, and four unopened petals that form a small central ball (Bullock and Harrison, 2005).
2.1 Scientific Classification of Syzygium aromaticum

Kingdom: Plantae
(Unranked) Eudicots
(Unranked) Roside
Order:                          Myrtales
Family: Myrtaceae
Genus: Syzygium
Species: S. aromaticum
2.2 Description of Cloves
Cloves are the aromatic dried buds of a tree (Syzygium aromaticum) used as a spice in virtually all the world's cuisine. The term 'Clove' is derived from the French word 'Clou' and the English word 'Clout', both meaning 'nail'- from the likeliness of the flower bud of the Clove tree to a broad headed nail. The Clove tree is an evergreen tree, which grows to a height ranging from 8-12m, having large square leaves and sanguine flowers in numerous groups of terminal clusters. The flower buds are at first of a pale color and gradually become green, after which they develop into a bright red, when they are ready for collecting. Cloves are harvested when 1.5-2 cm long, and consist of a long calyx, terminating in four spreading sepals, and four unopened petals, which form a small ball in the center (Alqareer et al,2012).
The seeds should be collected from fully ripe fruits for raising seedlings. Fruits for seed collection known commonly as "mother of clove" are allowed to ripe on the tree and drop down naturally. Such fruits are collected and sown directly in the nursery or soaked in water overnight and the pericarp removed before sowing. The second method gives quicker and higher percentage of germination. Only fully developed and uniform sized seeds, which show the signs of germination by the presence of pink radicle, are used for sowing. It is advisable to sow the seeds immediately after harvest. Heaping the fruits or keeping them tied up in air tight bags hastens the death of the seeds. Beds of 15-20 cm height, 1m width and conventional length are prepared for sowing seeds. The fertilizers must be applied in two equal split doses during the months of May-June and September-October in shallow drenches dug around the plant about 1 -11/m away from the base Harvesting and processing. The trees begin to flower in 6 years. Full bearing is achieved by about 20 years and the production continues for 80 years or more. Bearing between years shows much variation. Clove clusters are handpicked, when the buds reach full size and turn pink but before they open. At this stage, they are less than 2 cm long. They are spread thinly on mats and stirred frequently for uniform drying. Well dried cloves will snap cleanly with a sharp click across the thumb nail and weigh about one third of the green weight. The opened flowers are not valued as a spice. Harvesting has to be done without damaging the branches, as it adversely affects the subsequent growth of the trees. On an average, a clove tree yields 3.5-7.0 kg/year, depending upon the age, size and condition of the tree (Alqareer et al, 2012).
2.3 Climate and Soil
Clove trees grow well in rich loamy soils of the humid tropics and can be grown successfully in the red soils of the midlands of Kerala as well as in the hilly terrain of Western Ghats at higher elevations in Tamil Nadu and Karnataka. A cooler climate with well distributed rainfall is ideal for flowering; it thrives well in areas receiving an annual rainfall of 150-300 cm. The site selected for cultivation of clove needs good drainage, since crop cannot withstand water logging (Gulcin et al., 2012).
2.4 Nutrient Content of Cloves
The composition of the clove varies according to the agro climatic conditions under which it is grown, processed and stored. The dried clove bud contains carbohydrates, fixed oil, steam-volatile oil, resins, tannins, proteins, cellulose, pentosans and mineral elements. Carbohydrates comprise about two-thirds of the weight of the spice. The dried dark and flower buds also contain nutrients like proteins, minerals, vitamins, etc (Gulcin et al, 2012).
2.5 Chemical Constituents

Clove comprises of volatile as well as non-volatile constituents. Volatile Constituents Clove yields different types of volatile oil [oil extracted from i. leaves, ii. the stem, iii. the buds and iv. the fruit. These oils differ considerably in yield and quality. The yield and composition of the oil obtained are influenced by its origin, season, variety and quality of raw material, maturity at harvest, pre- and post-distillation treatments and method of distillation. The chief component of all the types of oil is eugenol (Gulcin et al, 2012). The compound eugenol is responsible for most of
the characteristic aroma of cloves Eugenol composes 72–90% of the essential oil extracted from cloves and is the compound most responsible for clove aroma. (Kamatou et al, .2012). 100% extraction occurs at 80 minutes in pressurized water of 125°C. (Rovio et al, .2000) Ultrasound-assisted and microwave-assisted extraction methods provide more rapid extraction rates with lower energy costs. (Khalil et al,.2017) Other important essential oil constituents of clove oil include acetyl eugenol, beta- caryophyllene and vanillin ,crategolic acid , tannins such as bicornin, (Kamatou et al,.2012)(Li-ming et al,.2012) gallotannic acid , methyl salicylate (painkiller), the flavonoids eugenin , kaempferol , rhamnetin , and eugenitin , tri terpenoids such as oleanolic acid ,stigmasterol, and campesterol and several sesquiterpenes(Dan benskey et al,. 2004)

2.5.1   Bud Oil

Good-quality clove buds contain 15-20% essential oil (Gopalakrishnan et al., 1988). The oil is dominated by Eugenol (70-85%), eugenyl acetate (15%) and p-caryophyllene (5-12%), which together make up 99% of the oil. The constituents of the oil also include methylamylketone, methyl salicylate, a-and p-humulene, benzaldehyde, p-ylangene and chavicol. The minor .connstituents like methylamylketone, methylsalicylate etc., are responsible for the characteristic Pleasant odour of cloves. The clove bud and stem oils from Madagascar were also dominated by eugenol, eugenyl acetate and p-caryophyllene. The stem oil contained a higher level of eugenol, whereas the eugenyl acetate content was higher in the bud oil. The oil from clove bud contained 73.5-79.7% eugenol and 4.5- 10.7% eugenyl acetate, while the stem oil contained 76.4-84.8% eugenol and 1.5-8.0% eugenyl acetate. Both contained 7.3-12.4% p-caryophyllene and 1.0-1.4% a-humulene3 Pino et al. identified 36 compounds from the volatile oil of clove buds. Clove buds from India contained 12.9-18.5% oil, of which 44-55% was eugenol, whereas the pedicels contained 3.0-7.7% oil with 60.0-72.4% eugeno!2. Leaf Oil Clove leaves yield 3.0-4.8% essential oil. The essential oil content during the different stages of leaf growth revealed that the eugenol content in the leaves increased from 38.3 to 95.2% with maturity, while the contents of eugenyl acetate (51.2 to 1.5%) and caryophyllene (6.3 to 0.2%) decreased 4. Clove bud and leaf oil contain various classes of compounds, e.g. monoterpenes, sesquiterpenes, aldehydes and ketones. Clove Stem Oil Clove stem yields 6% volatile oill. The oil is a pale to light yellow liquid containing 80.2% eugenol and 6.6% P-caryophyllene, besides several minor components. Fruit Oil Ripe fruits yield 2% of oil, which is comprised of 50-55% eugenol (Gopalakrishnan et al, 1988)
2.5.2     Non-volatile

 Constituents A few non-volatiles have been isolated from clove, which include tannins, sterols, triterpenes and flavonoids. (Gopalakrishnan et al.1988)
2.5.3 Tannins
Cloves contain 10-13% tannins, which have the same chemical composition as gallotannic acid. Eugenin and ellagitanninS were isolated from cloves. Eugenolglucosidegallate, a chromone C-glycoside, galloyl and hexahydroxydiphenyl esters of 2, 4, 6-trihydroxy acetophenone- 3-ducopyranoside were isolated from clove Ieaves6. Further, two ellagitannins, namely, syzyginin A (1, 2, 3-tri-O-galloyl-4, 6-(S) - tergalloyl-p-D-glucoside) and syzyginin B, were also isolated from the leaves. Triterpenes Cloves contain about 2% of the triterpene, oleanolic acid. Narayanan and Natu (1974) isolated maslinic acid from clove buds?. From clove, 2oc-hydroxyoleanolic acid was also isolated. SterolsSterols isolated from clove include sitosterol, stigmasterol and campesterol. (Lai and Roy,  2004).
2.5.4 Flavonoids
A chromone  C-glucoside, isobiflorin (5,7-dihydroxy-2-methoxychromone-8-C-p-D-glucopyranoside) and biflorin were isolated from the ethanolic extract of cloves9. From the ethanol extract of the seeds, apigenin 6-C-[p-D-xylopyranosyl-(l—>2")- (3-Dgalactopyranoside]-7-O-p-D-glucopyranoside and apigenin-6-C-[|3-D-xylopyranosyl- (1—* 2")-[3-Dgalactopyranoside]-7-O-p-D-(6-0-pcoumarylglucopyranoside) were isolated (Lai and Roy, 2004).
2.6 Medical Uses
Clove is known to possess antibacterial properties and is used in various dental creams, tooth pastes, mouth washes, and throat sprays to cleanse bacteria. It is also used to relive pain from soregums and improves overall dental health. In dentistry, eugenol in combination with zinc oxide is used for temporary filling of cavities. Clove is an anodyne (an agent that soothes or relives pain) for dental emergencies. Cloves are aphrodisiac (an agent for arousing or increasing sexual desire or potency). Clove is used as an anti-inflammatory agent, due to its high content of flavonoids. Aroma therapists use pure clove oil to cure the symptoms of rheumatism and arthritis. Clove is used as a carminative, to increase hydrochloric acid in the stomach and to improve peristalsis. Apply the paste of clove powder in honey to treat acne. Paste of clove powder in water promotes faster healing of cuts and bites. Cloves can effectively cure many digestive problems. It is having medicinal qualities to cure flatulence, loose motions, indigestion and nausea. (Pandey and Singh,2011). Cloves are useful in relieving the symptoms of diarrhea, gastric irritability and vomiting. Clove and clove oil boost the immune system by purifying the blood and help to fight against various diseases. Clove oil is effective in curing Athlete's foot and nail fungus. Cloves are good expectorants that promote the discharge of mucous and secretions in the respiratory passage. The aromatic clove oil, when inhaled can help soothe certain respiratory conditions like cold, cough, asthma, bronchitis, and sinusitis. It also helps in clearing the nasal tract. Cloves can effectively prevent the lung cancer as well as the skin cancer. Eugenol helps in minimizing the harmful effects of environmental wastes that can cause cancer of digestive system. Clove oil stimulates blood flow and circulation making it useful for the people having cold extremities.
Cloves benefits the diabetic patients by controlling the blood glucose levels. Eugenol is powerful enough for preventing blood clots. Sucking of a clove bud reduces desire for alcohol. Muscular cramps are often relieved, when the oil of clove is applied as a poultice near the affected area. Cloves also help prevent the breakdown in retina of the eye, which slows down macular degeneration and aids vision in the old age.  The underlying mechanism is through the prevention of the breakdown of docosahexaenoic acid, which preserves vision in elderly people (Pandey and singh, 2011). Researchers found that sniffing the spicy aroma of cloves reduces drowsiness, irritability and headaches. One drop of clove oil applied to the roof of the mouth can instantly relieve many headaches. Clove enhances memory retention. It is recommended for relieving brain fog, lethargy and depressive state of mind. Research has shown that clove oil is an effective mosquito repellent. Clove may be looked upon as the champion of all the anti-oxidants known till date. The Oxygen Radical Absorption Capacity test (ORAC) is a scale developed by U.S. Department of Agriculture for comparing antioxidant activity. The ORAC score, of clove is over 10 million. A drop of clove oil is 400 times more powerful as an anti-oxidant than wolf berries or blueberries.
2.7      Traditional Medicinal Uses
Cloves are used in India Ayurvedic medicine, Chinese medicine and western herbalism where the essential oil is used as an anodyne (pain killer) for dental emergencies. Cloves are used as carminative, to increase hydrochloric acid in the stomach and to improve peristalsis. Clove are said to be natural anthelminitic (Balch et al,.2000). The essential oil is used in aromatherapy when stimulation and warming are needed, especially for digestive problems. Topical application over the stomach or abdomen are said to warm the digestive tract. Applied to a cavity in a decayed tooth, it also relief toothache (Alqareer et al., 2012).
In Chinese medicine, cloves or ding xiang are considered acrid. In addition, clove oil is used in preparation of some toothpastes and clovacaine solution, which is a local anesthetic used in oral ulceration and inflammation. Clove oil is mixed with zinc oxide to form a temporary tooth cavity filling (Youngken,1950).
2.8 Description of Staphylococcus aureus
Staphylococcus aureus is a Gram-positive , round-shaped bacterium that is a member of the Firmicutes ,and it is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin . It is often positive for catalase and nitrate reduction and is a facultative anaerobe that can grow without the need for oxygen. (Masalha et al,.2001). Although S. aureus usually acts as a commensal of the human microbiota it can also become an opportunistic pathogen, being a common cause of skin infections including abscesses ,respiratory infections such as sinusitis , and food poisoning . Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins , and the expression of a cell-surface protein that binds and inactivates antibodies .The emergence of antibiotic-resistant strains of S.aureus such as methicillin-resistant S. aureus (MRSA)is a worldwide problem in clinical medicine . Despite much research and development , no vaccine for S. aureus has been approved.Staphylococcus aureus on basic cultivation media. Hemolysis on blood agar, DNase activity, clumping factor, latex agglutination, growth on mannitol-salt and Baird-Parker agar, hyaluronidase production .An estimated 20% to 30% of the human population are long-term carriers of S. aureus (Kluytmans et al.,1997)(Tong et al.,2015)which can be found as part of the normal skin flora , in the nostrils, (Kluytmans et al.,1997)(Cole et al.,2001) and as a normal inhabitant of the lower reproductive tract of women. (Senok et al., 2009)(Hoffman and Barbara 2012) S. aureus can cause a range of illnesses, from minor skin infections,such as pimples, [] impetigo , boils , cellulitis, folliculitis,carbuncles, scalded skin syndrome , and abscesses, to life-threatening diseases such as pneumonia,meningitis, osteomyelitis, endocarditis , toxic shock syndrome , bacteremia , and sepsis . It is still one of the five most common causes of hospital-acquired infections and is often the cause of wound infections following surgery . Each year, around 500,000 patients in hospitals of the United States contract a staphylococcal infection, chiefly by S. aureus. (Bowersox and John,1999) Up to 50,000 deaths each year in the USA are linked with Staphylococcus aureus infections.(Schlecht et al.,2015)

2.8.1 Scientific Classification of Staphylococcus aureus
Domain:  Bacteria
Phylum:  Firmicutes
Class:  Bacilli
Order:  Bacillales
Family: Staphylococcaceae
Genus: Staphylococcus
Species: S. aureus
2.9 Description of Escherichia coli

Escherichia coli (coli,2005) also known as E. coli (wells, 2000) is a Gram-negative ,facultative anaerobic , rod-shaped , coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms(endotherms). (Tenaillon et al., 2010)(Singelon,1999). Most  E. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination .(E.coli 2012)(Volt and Dippold,2005). The harmless strains are part of the normal microbiota of the gut , and can benefit their hosts by producing vitamin K 2 , (Bentley and Meganathan,1982) and preventing colonization of the intestine with pathogenic bacteria, having a symbiotic relationship. (Hudult et al., 2001)(Reid et al, .2001) E. coli is expelled into the
environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for 3 days, but its numbers decline slowly afterwards. (Russell and Jarvis, 2001). E. coli and other facultative anaerobes constitute about 0.1% of gut microbiota , (Eckburg et al,.2005) and fecal–oral transmission is the major route through which pathogenic strains of the bacterium cause disease. Cells are able to survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental samples for fecal contamination(Feng etal,.2002)(Thompson and Andrea,2007)A growing body of research, though, has examined environmentally persistent E.coli which can survive for extended periods outside a host. (Ishii and Sadowsky,2008).The bacterium can be grown and cultured easily and inexpensively in a laboratory setting, and has been intensively investigated for over 60 years. E. coli is a chemoheterotroph whose chemically defined medium must include a source of carbon and energy. (Tortora and Gerard, 2010) E.coli is the most widely studied prokaryotic model organism, and an important species in the fields of biotechnology and microbiology, where it has served as the host organism for the majority of work with recombinant DNA. Under favorable conditions, it takes up to 20 minutes to reproduce. (Bacteria, 2014).

2.9.1 Scientific Classification 0f Escherichia coli

Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: E. coli

                                               CHAPTER THREE
3. 1 Sample Collection
A dried sample of cloves (Syzygium aromaticum) is collected from central market birnin kebbi Kebbi state. The plant was identified authenticated by a taxonomist in botany department
3.2 Preparation of the Plant Extract
The plant material cloves (Syzygium aromaticum) is collected from central market birnin kebbi, by observing it physical characters like shape, smell and colour of the plant. The sample is then pound to powder with the help of mechanical grinder i.e mortar and pestle and then stored in a plastic sterilized container.
        50g of the powdered plant was weighed and diluted in 500ml of sterile water in a conical flask sealed with a sterile foil paper and was kept for 24hours and allowed for proper diffusion, the mixture is then filtered with a clean muslin cloth and filter paper. The liquid is taken to the water bath for proper concentration. (Alqareer et al. 2012)
3.3 Collection of Test Organism
The pure isolate Staphylococcus aureus and Escherichia coli were collected from Kebbi University Of Science and Technology Aliero Laboratory and were sub-cultured using nutrient agar for S.aureus and Mueller Hilton agar for E.coli to obtain a pure culture and were inoculated using streaking method.the plates were labeled respectively and then incubated at 37c to obtain the viable culture of the susceptibities of the test organisms.

3.4 Qualitative Phytochemical Analysis
The extract of the plant is to be screened for the presence of various secondary metabolites (phytochemical) such as flavonoids, tannins, saponins, glycosides, and Terpenoids
3.4.1 Test for Tannins
Aqueous extract is boiled in a water bath in the sterile test tube and 0.1 % of ferric chloride will be added and observe for brownish green or blue coloration. (Cappuccino and Sherman, 2014)
3.4.2 Test for Flavonoids
A 5ml of dilute ammonia is added to a portion of the aqueous filtrate of each plant Extract by the addition of concentrated H2SO4 is added and a yellow coloration is obsen'ed which indicate the presence of flavonoids. (Cappuccino and Sherman. 2014)

3.4.3 Test for Saponins
A 10ml filtrate is mixed with 5ml distilled water and shacked vigorously for a stable persistent growth. Three (3) drops of olive oil is to be added and shacked vigorously, then observed for formations of emulsion. (Cappuccino and Sherman, 2014)

3.4.4 Test for Glycosides

A 0.2g of the extract will be weigh and 2ml of sulphuric acid is to be added and boiled for 15min after which 1ml of Fehling solution is added and brick red coloration is observed. (Cappuccino and Sherman, 2014)
3.4.5 Test for Phenol A  fraction of the extracts will be treated with aqueous 5% ferric chloride and observed forformation of deep blue or black color. (Cappuccino and Sherman, 2014)
3.4.6 Test for Terpenoids

A 5ml of solution of the extract was added in a test tube. Then 2ml chloroform and 3ml concentrated sulphuric acid solution was added, a reddish brown precipitate was formed between upper and lower layer of the solution.(El-oley et al.,1994)
3.5 Preparation of Mueller Hilton agar

This was prepared in accordance with the manufacturer’s instruction. 37g of Mueller hilton agar powder was dissolved in 1000ml of distilled water in a conical flask. This was heated on a hot plate for proper dissolution, after which the flask was corked and autoclaved at temperature of 1210c for 15min. the flask was removed and allowed to cool; 15ml were dispense in to sterile petri-dishes and allowed to solidify.
3.6 Preparation of nutrient agar

28gram of nutrient agar powder was weighed using a weighing balance and transferred into a conical flask containing 1000ml of distilled water. This was then heated on hot plate to completely dissolve the solution, and autoclaved at 1210c for 15min. after sterilization, the medium was allowed to cool at 400c, 10ml were poured in sterile maccartney bottles and kept in a slanting position. The slant was stored in a refrigerator.
3.7 Sensitivity Test Using Agar well diffusion method

The antibacterial activity of the clove extract was determined using agar well diffusion method. Mueller hilton agar was inoculated with the giving microorganisms i.e Staphylococcus aureus and Escherichia coli  into four(4) different agar plates by spread plate method and was labelled respectively. Well were punched using punch-borer (12mm) in the agar and each was loaded with the crude extract of the plant and the phytochemical compounds of the extract. Control wells containing neat solvent and ciprofloxacin were also loaded in the same plate. The plate were allowed to stand for 15min on the bench to allowed free diffusion of the extract to take place and then incubated at 370c for 24hours and antibacterial activity was access by measuring the diameter of the zone of inhibition (Gerhard).The zone of inhibition was measured and recorded using a transparent meter rule.

3.8   Minimum Inhibitory Concentrations and Minimum Bacteriocidal Concentration
MIC are the lowest concentrations of antimicrobial that will inhibit microbes after incubation overnight, while MBC are the lowest concentrations of antimicrobial that prevent the growth of organism after subculture on to antimicrobial free media. All can be done using dilution tube
method:-  Cloves was diluted into various concentrations, and 50mg/ml, in sterile
nutrient broth in test tubes. Using a wire loop 0.5 McFarland standard was Inoculated into test tubes containing 1 ml of the various concentrations of clove in nutrient broth. The tubes were incubated at 37°C for 18 to 24 hours and there after observed for growth or turbidity. Subsequently a loop full of broth from each test tube not showing growth, was inoculated into nutrient agar plate. Thereafter, equal volumes of sterile nutrient broth were added into the test tubes cultures and incubated further for 24 hours at 37°C. Then the tube and agar plate were examined for growth or turbidity using unaided eyes (CLSI, 2012).

                                               CHAPTER FOUR
4.1 Phytochemical properties of plant extract of cloves (Syzygium aromaticum)
The result of the phytochemical analysis of the plant extract of cloves (Syzygium aromaticum) showed the presence of  flavonoids, tannins, saponins, glycosides, phenol and terpenoids (Table 4.1)
4.2 Antibacterial Activity
 The antibacterial activity of clove extract using agar well diffusion method was indicated by zone of inhibition of 20mm by flavonoids. 5mm by tannins, 4mm by saponins,15mm by glycosides,2mm by phenol and also 16mm by terpenoids in Staphylococcus aureus and 16mm by flavonoids, 2mm by tannins, 3mm by saponins 19mm by glycosides, 2mm by phenol and 5mm by terpenoids in Escherichia coli concentration levels is shown in Table 4.2, This indicates that the zone of inhibition depends on the concentrations of the extract, i.e the higher the concentration the larger the zone of inhibition and also the lower the concentration the lesser the zone of inhibition. this means that Staphylococcus aureus has higher zone of inhibition at flavonoids ,glycosides and terpenoids and Escherichia coli has higher zone of inhibition in glycosides ,terpenoids and flavonoids also. The Minimum inhibitory concentrations of the extract that will be able to inhibit or reduce the growth of the organism is 20mg/ml for the test organism and result is shown in table 4.5. Also the Minimum bacteriocidal concentrations of the extract that will be able to kill the organism is 20mg/ml in Table 4.3.

Extract               Flavonoids     Tannins     Saponins     Glycosides     Phenol      Terpenoids

Table 4.1 Qualitative Phytochemical Screening of Cloves (Syzygium aromaticum)

Cloves                   ++                 +                    ++                   ++               +                   +++

+: present in small amount\concentrations
++: present in mild concentrations
+++: present in high concentrations

Table 4.2 Sensivity Test  of  Staphylococcus aureus
_______________________________________________________________________                 Isolate various concentration/ zone of inhibition in mm               _______________________________________________________________________
Staphylococcus Flavoniods   Tannins   Saponins   Glycosides   Phenol   Terpenoids

20mm             5mm      4mm        15mm     2mm       16mm
Extract: 10mm
Control: Ciprofloxacin  22mm

Table 4.3 Sensitivity Test of  Escherichia coli
Isolate                                             various concentration\zone of inhibition in mm

Escherichia coli         Flavaniods  Tannins  Saponins  Glycosides  Phenols Terpenoids

                                  16mm       5mm         3mm       19mm         2mm       18mm
Extract: 5mm
Control: Ciprofloxacin 20mm

Table 4.4 Minimum Inhibitory Concentration (MIC) And Minimum Bactericidal Concentration (MBC) of Cloves
Organism (MIC) (MBC)
S. aureus


+Means Turbidity/Growth in ager plate
-Means no Turbid/Absent of growth in ager plate


                                                       CHAPTER FIVE
  This research showed that Clove extract (S. aromaticum) contains flavonoids, tannins, saponin, glycoside, phenol, and terpenoids (Table 4.1). The test showed that the higher the concentrations of the extract the higher the zone of inhibition with the diameter of 20mm, 16mm, 15mm, 5mm, 4mm and 2mm in Staphylococcus aureus and 19mm, 18mm, 16mm, 5mm in Escherichia coli at different concentration levels of phytochemical constituents respectively. The minimum inhibitory concentration shows highest effectiveness as 20mg/ml and the minimum bactericidal concentration shows the lowest effectiveness of l0mg/ml.
Clove extract was the most effective as it showed more inhibition of the test bacteria   which was compared with that of standard drugs. Clove extract displayed significant inhibition of   the test bacteria which was compared with that of standard drugs. Clove extracts displayed significant activity against the entire test organism as standard activities. Different phytochemicals have been found to possess a wide range of activities, which may help in protection against chronic diseases for example alkanoid protect against chronic diseases, saponins protect against hypercholesterolemia and antibiotic properties. Phytochemical screening of clove extract used to study the presence of flavonoids tannin, saponin ,glycoside, phenol, and terpenoids have various medicinal values such as anti-inflammatory, antidiabetic and analgesic activities and for central nervous system activity. The importance of alkaloids, saponin and tannins in various antibiotics used in treating common pathogens strains has recently been reported by (Mensah 2008).
 Clove extract are known to posses some antimicrobial activities and are used in various food preparation as flavor enhancers and in herbal medicine (Arora and kaur 1999). their antimicrobial agent include thymol, terpenes, eugenol, flavones, glycoside of phenolic monoterpenoids and aliphatic alcohols among other elements (Deans and Ritchie, 1987). These substances acting alone or in combination may result in a broad spectrum activity for Streptococcus species and other bacteria. The World Health Organization (WHO, 2003) established that the daily quantity acceptable of clove per day is 2.5 mg/kg of weight in humans (Gulcin et al, 2012).
5.1 Conclusion

  From this research, Clove was active against staphylococcus aureus was found to be 20mm, 16mm, 15mm, 5mm, 4mm and 2mm zone of inhibition and Escherichia coli was found to be 19mm, 18mm, 16mm, 5mm, 3mm,and 2mm respectively. The minimum inhibitory concentration (MIC) was 20mg/ml, and the Minimum bacteriocidal concentration was l0mg/ml. Phytochemical components determined are flavonoids, Saponins, Phenols, Tannin, Glycoside and terpenoids.
5.2 Recommendation
I recommend that, clove should be used at times  instead of consumption of a lot of antibiotic drugs which may lead to resistance if taken excessively. The fact that the extract of these medicinal plants inhibited some medically important bacteria proves that the plant might have some potential as an alternative source of antibacterial substances.

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