ABSTRACT
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.
TABLE OF CONTENTS
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
CHAPTER 2: LITERATURE REVIEW
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
CHAPTER 3: MATERIALS AND METHODS
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
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Results 44
4.2 Discussion
54
CHAPTER 5: CONCLUSION AND
RECOMMENDATIONS 59
5.1 Conclusion 59
5.2 Recommendations 60
References 61
Appendices 82
LIST OF TABLES
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
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
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.
1.2 STATEMENT OF PROBLEM
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.
1.3 JUSTIFICATION
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).
1.5
OBJECTIVES OF THE STUDY
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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