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The use of antibiotics in poultry production has been implicated in the high level of resistance to antibiotics among Avian pathogenic Escherichia coli (APEC) isolates. Medicinal plants with reported antibacterial properties are promising alternatives to the use of antibiotics in poultry production. The aim of this study was to investigate the antibacterial activities of selsected medicinal plants (Thevetia nerifolia, Zingiber officinale, Asystecia giganticum, Alchornea cordifolia, Dalium guineense) against APEC isolated from poultry farms in Umuahia and Afikpo. Forty eight (48) E. coli isolates were obtained from 46 faecal and 20 entrail samples obtained from 12 poultry farms. Twenty (62.5%) of the isolates were positive to in-vitro (Congo red) and in-vivo pathogenicity (day old chicks lethality) test which were designated as APEC. The antibiotic susceptibility testing of the APEC isolates to common antibiotics was carried out. The antibacterial activity of the extracts of the medicinal plants was investigated against the APEC isolates and a type culture of APEC. Percentage resistance of the APEC isolates to routine antibiotics was in the order, Ampicillin (100%), Cephalexin (95%), Pefloxacin (85%), Nalidixic acid (80%), Augmentin (80%), Streptomycin (80%), Septrin (70%), Gentamycin (65%), Ciprofloxacin (65%)  and Ofloxacin (35%). The isolates showed multiple antibiotics resistance (MAR) with MAR Index in the range of 0.3 to 1.0 and accompanying frequencies range of 1.0 to 5.0. The antibacterial susceptibility test of the plant extract showed that T. nerifolia and Z. officinale had no antibacterial activity against the APEC isolates while A. giganticum, A. cordifolia and D. guineense possessed some antibacterial activity with the highest significant (p<0.05) activity obtained with A. giganticum (17.76mm). The antimicrobial activity of the plant extracts was in the order: Asystecia giganticum>Alchornea cordifolia >Dialium guineense. Their MIC was noted as 250mg/ml. the photochemical screening of the plant extracts using semi-quantitative (chemical) method showed the varying presences of alkaloids, tannins, phenols, steroid, terpenoid and glycosides. The study reveals that Alchornea cordifolia, Asystecia giganticum and Dialium guineense possess antibacterial activity against APEC and could be a possible source of antimicrobial agents for use on the poultry farms.



Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              ix

Abstract                                                                                                                      x                                                                                                                                 

CHAPTER 1: INTRODUCTION                                                             

1.1       Background of the Study                                                                               1

1.2       Statement of problem                                                                                     3

1.3       Justification                                                                                                     4

1.4       Aim of the study                                                                                             9

1.5       Objectives of the study                                                                                   9



2.1       Medicinal Plants as Antimicrobial Agents                                                      10

2.2       Plant Phytochemicals and Antimicrobial Activities                                       11

2.2.1    Alkaloids                                                                                                         11

2.2.2    Phenolics                                                                                                        12

2.2.3    Terpenoids and essential oils                                                                          19

2.3       Extraction of Plants Phytochemical                                                               21

2.4       Antimicrobial Assay of Plant Extracts                                                          24

2.5       Escherichia coli                                                                                              26

2.5.1    Extraintestinal pathogenic E. coli (ExPEC).                                                  28

2.5.2    Avian pathogenic E. coli (APEC)                                                                  30

2.5.3    Isolation of E. coli from bird material                                                31

2.5.4    Methods of designation of E. coli as APEC                                                  31

2.5.5    Similarities between human ExPEC and APEC                                             34

2.6       Antibiotics Resistance and E. coli                                                                  35



3.1       Study Area                                                                                                      37

3.2       Collection and Preparation of Leaf Samples                                                  37

3.3       Extraction of leaves                                                                                        38

3.4       Phytochemical Analysis of the Leaves                                                           38

3.4       Sampling.                                                                                                        38

3.5       Isolation of E. coli                                                                                          39

3.6       Identification of isolates                                                                                 39

3.7       In Vitro Pathogenicity Testing                                                                        40

3.8       In Vivo Pathogenicity Testing (Confirmation Test)                                        40

3.9       Determination of Susceptibility Pattern of Isolates to Routine Antibiotics   41

3.10     Reconstitution of extracts                                                                               42

3.11     Determination of in-vitro Antimicrobial Activity of Plant Extracts               42

3.12     Determination of Minimum Inhibitory Concentration (MIC) of the

Extracts                                                                                                           43

3.13     Statistical Analysis                                                                                          43



4.1       Results                                                                                                            44

4.2       Discussion                                                                                                       54


5.1       Conclusion                                                                                                      59

5.2       Recommendations                                                                                          60

References                                                                                                      61

Appendices                                                                                                     82






2.1:      Main groups of plant compounds with antimicrobial activity                       20

4.1:      Frequency of isolation of APEC from sample sources                                   47

4.2:      Percentage susceptibility of APEC isolates to individual antibiotics             48

4.3:      Frequency of multiple antibiotics resistance (MAR) of APEC isolates to    

the tested antibiotics.                                                                                      49

4.4:      Diameter zone of inhibition of methanolic extracts of medicinal plants

against APEC Isolates.                                                                                   49

4.5:      Comparison of antimicrobial activities between medicinal plant extracts     50

4.6:      Minimum inhibitory concentration of the selected plants to standard APEC

            strain (ATCC 11175)                                                                                      51

4.7:      Classes of compounds found in the methanolic extracts of selected plants   52








Recently, there has been a widespread global report of antibiotic resistance in Escherichia coli (E. coli) (Ramirez and Tolmasky, 2010; King et al., 2012; Barlett, Gilbert and Spellberg 2013; Liu et al., 2016). This has not only complicated treatment but has also resulted to high cost of treatment. The abundance of these antibiotic resistant E. coli may act as a major reservoir for resistance genes for the spread of resistance within a community (Sengupta et al., 2013). Thus, severely limiting therapeutic options available for human and animal infections and ultimately resulting in high morbidity and mortality (Rice, 2009).

Escherichia is Gram-negative, rod-shaped, facultative anaerobic coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms) (Tenaillon et al., 2010). It utilises wide variety of substrates and has optimum growth temperature of 37°C (98.6°F). Being a facultative anaerobe, it is able to survive in difficult environments. E. coli is able to give out its DNA material through horizontal mechanisms such as transduction or conjugation resulting in new progenies or modification of an old population. This allows for a huge diversity in the organism which is expressed both phenotypically and genetically so much that the overall similarity of genes among all strains is only about 20% (Lukjancenko et al., 2010).

Escherichia coli can be majorly grouped into two namely: commensals (non-pathogenic) and non – commensals (pathogenic). The pathogenic group is divided into two other subgroups, known as Diarrheagenic E. coli (DEC), (these are obligate pathogens often implicated in gastrointestinal diseases) and Extraintestinal pathogenic E. coli (ExPEC) (facultative pathogens found in the gut of  few healthy individuals and animals as commensals but possesses the ability to invade other body organs and establish infection). The DEC pathotype is made up of eight subpathotypes which includes: Diffusely adherent E. coli (DAEC), Enteropathogenic E. coli (EPEC), Enterohaemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC),  Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC),  Shiga-toxin producing enteroaggregative E. coli (STEAEC) and Adherent invasive E. coli (AIEC) (Huang et al., 2006; Clements et al., 2012). The ExPEC pathotype is divided into six other subpathotypes: Avian pathogenic E. coli (APEC), Uropathogenic E. coli (UPEC), Sepsis/newborn meningitis associated E. coli (NMEC), Sepsis-associated pathogenic E. coli (SePEC), Mammary pathogenic E. coli (MPEC), and Endometrial pathogenic E. coli (EnPEC) (Mokady et al., 2005; Shpigel, Elazar and Rosenshine, 2008; Sheldon et al., 2010; Kunert et al., 2015). ExPEC has been implicated in zoonotic transmission (Cortes et al., 2010).

ExPEC has a substantial morbidity and mortality; with mortality rate of approximately 80% owning to increasing multidrug resistance among the strains, and has so become a global challenge (Ron, 2010; Schmiemann et al., 2012). It has a worldwide distribution and the potential to invade many tissues and cause infection in any age group (Turhan et al., 2015). ExPEC strains are associated with infections particularly common in both humans and poultry (Lutful, 2010). The strains have been found to cross the species barrier and effectively colonize humans as well as other animals (Johnson et al., 2009 Tivendale et al., 2010; Chanteloup et al., 2010). Many possible reservoirs for ExPEC have been identified through molecular epidemiology studies from around the world, and these possible reservoirs include: the human gut, pets, food animals, retail meat products, sewage and other sources from our sorroundings (Manges and Johnson, 2012). Studies indicates that poultry is the animal food source most closely linked to human ExPEC (Barbieri et al., 2015). This is because Poultry meat has shown a maximal amount of E. coli contamination, with more extensive antimicrobial resistance than E. coli recovered from other meat sources (Mellata, 2013; Mitchell et al. ,2015). There is also a huge similarity between the virulence genes of these E. coli isolates and that of human ExPEC (Barbieri et al., 2015). Thus, giving rise to speculations that it has the ability to cause diseases as well. It is believed that Human-associated ExPEC evolved from; or are the same as Avian pathogenic E. coli (APEC), since both possess same pattern of antimicrobial resistance and conversely has the potential to interchangeably cause infection in their various hosts.



Avian pathogenic E. coli (APEC) is a pathosubgroup of ExPEC afflicting birds. The APEC isolates are reportedly becoming progressively more resistant to antibiotics (Barbieri et al., 2013; Carvalho et al., 2015; Koga et al., 2015).  It has been speculatively implicated in the emergence of antibiotic resistance strains in humans (Hannah et al., 2009; Johnson et al., 2012; Olsen et al., 2012). This resistance has been largely attributed to indiscriminate and unregulated use of antibiotics in animal husbandry, often in sub therapeutic doses to control microorganisms especially E. coli for the optimal growth of the animal (growth promoter) or used therapeutically for ever present outbreak of E. coli diseases (collibacillosis) (Ramirez and Tolmasky, 2010; King et al., 2012). Excessive exposure of commensals like E. coli to antibiotics increases the breed of resistant bacteria. And, if the resistance is plasmid-mediated as often is the case, resistance might be transferred to a more virulent bacteria, thus, making treatment of infection increasingly complicated by the emergence of resistant bacteria especially to most first-line antimicrobial agents (Ramirez and Tolmasky, 2010; Vanessa et al., 2014). The reservoir of resistant bacteria in food animals suggests a possible risk of dissemination of resistant bacteria, or resistance genes, from food animals to humans (Cortes et al., 2010). The implication of the use of antimicrobial drugs for growth promotion and therapeutic value in food animals in the breed of antibiotics resistance has received much attention. Thus, necessitates the need to find an alternative which will produce the desired results in farm with minimal or no side effects to humans. And plant antimicrobial is a promising prospect.



The use of medicinal plants to treat ailments has been in practice for as long as man and employed all over the world (Malini et al., 2013). Plants are known to contain phytochemical such as tannins, terpenoids, alkaloids and flavonoids which are responsible for their therapeutic activities (Okigbo et al, 2009). These phytochemicals are often constitutive in nature, or they are as a response to stimuli in the environment and often act in synergy to give the plant its therapeutic benefits.  The advent of drug resistance by bacteria has revived interest in research in medicinal plants as possible antimicrobial agents. Studies have demonstrated some of these plants as possessing antibacterial activities. Medicinal plants such as Thevetia nerifolia, Alchornea nerifolia, Dialium guineense, Asystecia gigantica and Zingiber officinale have been reported to have antibacterial activity especially against E. coli isolates.

Thevetia nerifolia, also called Thevetia peruviana, but commonly called exile tree: exile oil tree; milk bush (Irvine), lucky nut tree, trumpet flower and olómiòjò in Yoruba is an evergreen tropical shrub that belongs to the family Apocynaceae (Bandara et al., 2010). Its leaves are willow – like, linear, lanceolate and glossy green in colour. It is a plant that is opportunistic in nature and thrives well in moist sandy soil but also tolerates other soil, sometimes cultivated as an ornamental or hedge that blooms throughout the year (Bandara et al., 2010). The plant has great repute for its toxic properties other than its therapeutic value. It is particularly known for its Cardiac glycosides and other cytotoxic compounds such as Thevetin A and B, Thevetoxin, Peruvoside, Ruvoside and Nerifolin which have been investigated by researchers (Guptae et al.,2011; Naza et al., 2015). A controlled quantity of the plant extract is said to possess medicinal properties and thus the whole plant and its various parts have been employed in formulations as purgative, diuretic, cathartic and febrifuge to treat bladder stones, edema, ringworm, ulcers, dropsy, insomnia, leprosy, haemorrhage, intermittent fevers, ringworm, rheumatism, tumors etc (Guptae et al.,2011).

Alchornea cordifolia, commonly called the Christmas bush, ‘ubobo’ and evwa’ in Urhobo and Isoko languages of Delta State of Nigeria, also known by other names such as dovewood (Amos-Tautua et al.,2011).  It is a shrub distributed throughout tropical Africa and widespread in secondary forest. It likes marshy areas or riparian habitat but also spread into drier ecosystems, particularly disturbed soils. It grows up to 5,000 feet (1,524 m) in altitude and it is often planted in rows as windbreaks to protect other crops. It returns calcium to exhausted or poor soils. The plant occurs mainly in West to Central Africa in countries such as Congo, Ivory Coast, Nigeria and Ghana. A black dye is produced by the fruits which is traditionally used for dyeing of cloth, pottery, fishing nets and leather. Crafts and constructions are made with the wood, while in West Africa, the leaves are used for packing cola nuts and ‘okpeyi’, a Nigerian condiment produced by fermenting seeds of Prosopis Africana (Guill and Perr.) Taub (Mavar-Manga et al., 2007). Various parts of the plant are used in traditional African medicine as  enema,  cicatrizant to wounds,  painkiller, immune booster, to prevent miscarriage and treat various infections and diseases such as skin infections, venereal diseases, leprosy, sores, abscesses, yaws, filariasis, fevers, respiratory problems amoebic dysentery, diarrhoea and conjunctivitis (Fomogne-Fodjo et al., 2014; Owhe-Ureghe and Akpo, 2016). Research studies have shown the plant extracts to possess antibacterial activity against Helicobacter pylori, Salmonella typhi, Shigella flexneri Salmonella enteritidis, enterohemorrhagic E. coli (EHEC), antifungal and antiprotozoal properties as well as anti plasmodial activity (Osadebe et al., 2012).

Dialium guineensis also commonly known as velvet/black tamarind, tumble tree, black tumber, also called Awin or Igbaru in Yoruba, Icheku in Igbo and Tsamiyar kurm in Hausa, is a tall, tropical, fruit-bearing tree that belongs to the Leguminosae family. It is small, about grape size, with brown hard inedible shells. It grows in dense forests in Africa. It can be found in West African countries such as Ghana, Togo, Sierra Leone, Senegal and Guinea –Bissau. It reaches up to 30 metres in height with butt flares that are thin, narrow that grows up to 80cm in diameter. It has hard wood that is often used for construction, firewood and charcoal production. The fruit pulp is eaten soaked in water as a beverage or consumed raw. The different parts of the plant have medicinal properties and are traditionally used against several health conditions   such as bronchitis, diarrhoea, severe cough, wound, stomach aches, malaria fever, jaundice, haemorrhoids, genital infection, ulcer, for oral health and to improve lactation  (Ogu and Amiebenomo, 2012). The methanolic leaf and stem bark extracts have been reported to possess anti-vibrio and anti-diarrhoeal potentials (Ogu and Amiebenomo, 2012).

Asystasia gangetica commonly known as the Chinese violet, creeping foxglove or ganges primrose is a species of plant in the Acanthaceae family. It is a fast - growing perennial plant, shrub by herb which grows to 1 m height and has a remarkable tolerance for a vast array of habitats spanning from disturbed, semi-waterlogged to cultivated areas where it forms a dense ground cover. It is native in Africa and Asia and used as a forage for cattle, goats and sheep in South-East Asia.  A. gangetica is widely used in traditional medicine for the treatment of asthma, stomach-ache, snakebites, epilepsy, urethral discharge, rheumatism ulcers, dry coughs, analgesic during childbirth, cicatrizant for sores, wounds and piles, in embrocations for stiff neck and enlarged spleen, as a vermifuge, antihelmintic and inflammation and cancer (Tillo et al., 2012).   Studies have shown A. gigantica to possess antibacterial and antifungal properties (Hamid et al., 2013).  

Zingiber officinale commonly called Ginger and aje by Yorubas, jinja by Efiks/Ibibios of Cross River and Akwa Ibom States is a herbaceous perennial reed - like plant with annual leafy stems, about a meter (3 to 4 feet) tall plant that belongs to the family Zingiberaceae (Osabor et al., 2015). Ginger originated in the lush tropical jungles in Southern Asia in the wide but its aesthetic appeal and the adaptation to warm climates has made it cultivated and often used for beautification in the subtropics. Traditionally, the rhizome is gathered when the stalk withers; it is immediately scalded, washed and scraped to kill it and prevent sprouting.  The plant leaves and flowers are used medicinally for ailments such as stomach disorder, Rheumatism, diabetes, wounds, baldness, snake bite, toothache, arthritis, respiratory disorders, bleeding, rash etc (Mashhadi et al.,2013; Liu et al.,2013; Manosroi et al.,2013; Ribel–Madsen et al., 2015). Studies have also shown its antibacterial activity against gram negative bacteria like E. coli and its activity has been linked to Gingerol and other similar compounds (Islam et al., 2014).

The abundance of the medicinal plants in a tropical country such as Nigeria and the reported positive results on the antibacterial activities of some of these plants give basis for a study on their antibacterial activities on Avian pathogenic E. coli. More so, as there is a dearth of information on their activities on this particular E. coli strain. Also, the implication of this strain in the breed of antibiotics resistance especially on the poultry farms necessitates the search for a possible alternative to the use of antibiotics against it on the farm. This search must first begin with ascertaining the claims that “it is becoming progressively more resistant to antibiotics”. The provision of plant antimicrobial as an alternative to conventional antibiotics will ensure a readily available treatment for APEC infections, reduce over dependence on antibiotics on the farms, halt the zoonotic transmission of resistant E.coli strain to man and in the long run reduce the incidence of antibiotic resistance in the communities.

1.4       AIM OF THE STUDY

To evaluate the antibacterial activity of some medicinal plant extracts against Avian Pathogenic Escherichia coli (APEC).



i.                    To isolate Avian pathogenic E. coli (APEC) from some commercial poultry farms.

ii.                  To determine the susceptibility pattern of the isolated APEC to routine antibiotics.

iii.                To evaluate the antibacterial activities of the extracts against the APEC isolates.

iv.                To conduct the phytochemical analysis of the selected plants.


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