ANTIBACTERIAL PROPERTY OF HUMAN EAR WAX AGAINST PATHOGENIC

BACTERIA ISOLATED WITHIN JUJA TOWN

RONALDO CHACHA

A Research proposal submitted in partial fulfilment of the requirements for the degree of
Bachelor of Science in Industrial Biotechnology of Jomo Kenyatta University of
Agriculture and Technology.

SEPTEMBER 2019
DECLARATION

I hereby declare that this research proposal is my original work and has not been presented
elsewhere for a degree award

Signature……………………… Date…………………….

CHACHA RONALDO RIOBA (HSB215-0151/2016)

Declaration by Supervisor

This Research Proposal has been submitted for examination with my approval as supervisor.

DR. STEVEN GER NYANJOM, Biochemistry Department, JKUAT

Signature……………………… Date………………….

ii
 TABLE OF CONTENTS
DECLARATION.......................................................................................................................................ii

TABLE OF CONTENTS.........................................................................................................................iii

LIST OF DIAGRAMS..............................................................................................................................v

LIST OF ABBREVIATIONS AND ACRONYMS................................................................................vi

ABSTRACT.............................................................................................................................................vii

CHAPTER ONE........................................................................................................................................1

1.0 INTRODUCTION.......................................................................................................................1

1.1 Background Information................................................................................................................1

1.2 Problem statement.........................................................................................................................2

1.3 Justification...................................................................................................................................2

1.4 Research questions........................................................................................................................3

1.5 Hypothesis....................................................................................................................................3

1.6 Study objectives............................................................................................................................3

1.6.1 General objectives..................................................................................................................3

1.6.2 Specific objectives..................................................................................................................3

CHAPTER TWO.......................................................................................................................................4

2.0 LITERATURE REVIEW...........................................................................................................4

2.1 Ear wax/ cerumen..............................................................................................................................4

2.2 Composition of cerumen....................................................................................................................4

2.3 Functions of ear wax..........................................................................................................................5

2.4 Antibacterial properties.....................................................................................................................6

2.5 Forms of cerumen..............................................................................................................................6

2.7 Mechanism of action of pathogenic bacteria......................................................................................8

2.8 Bacteria isolation...............................................................................................................................8

CHAPTER THREE...................................................................................................................................9

iii
 3.0 MATERIALS AND METHODS....................................................................................................9

3.1: Study area and sampling...................................................................................................................9

3.2 Study design and methods used.........................................................................................................9

3.2.1 Bacteria isolation and culturing..................................................................................................9

3.2.2 Bacterial inhibition test using cerumen.....................................................................................10

3.3 Data analysis....................................................................................................................................10

WORK PLAN..........................................................................................................................................11

BUDGET..................................................................................................................................................12

REFERENCES..........................................................................................................................................13

iv
LIST OF DIAGRAMS
Figure 2.2 showing dry earwax.......................................................................................................7
Figure 2.1 showing wet earwax.......................................................................................................7

v
LIST OF ABBREVIATIONS AND ACRONYMS

E. coli Escherichia coli

HBD1-3 Human Beta Defensins1-3

HNP Human Neutrophils Protein

HBP1 Human Bactericidal Permeably increasing protein

HSLPI Human Secretory Inhibitor

vi
 ABSTRACT
Ear wax is a product of the ear that protects the skin of the ear from water and infection, its
normally made of a yellowish waxy substance called cerumen secreted by apocrine ceruminous
glands in the outer canal. It can be used as an antibacterial agent. The main objective of the study
is to test for the antibacterial property of human earwax against pathogenic bacteria within Juja
town which will be isolated from meat samples, water samples and ‘dagaa’ samples. Different
composition of ear wax has been tested against different ear pathogenic strains and has proven to
possess antibacterial activity. The present work is aimed to test the antibacterial property and
inhibitory property of ear wax against the viability of pathogenic bacteria isolated within Juja
town. Bacteria isolated from three different sources within Juja town will be cultured to different
plates and serial dilution carried out to reduce a dense culture of cells to a more usable
concentration. The grown cultures will then be grown on various selective media to obtain a
sizeable number of pathogenic microbes to work with. Inhibitory property of ear wax will be
tested against the bacteria at different concentration at various percentages after carrying out
serial dilutions. Minimum inhibitory concentration will be determined on basis of our
concentration. Diameter of zones of inhibition will be measured to determine the extent of
antibacterial property of ear wax. Data obtained will then be collected into excel worksheets and
analysed by ONE-WAY ANOVA and results presented in pictures and tables.

vii
viii
 CHAPTER ONE
1.0 INTRODUCTION
1.1 Background Information
Ear wax also referred to as cerumen, is a rich biological fluid that has unique advantage as a bio
monitoring medium of a high diagnostic potential. Cerumen is formed at the ear canal by
sebaceous glands, ceruminous glands and apocrine glands present at the outer one third of the
human external auditory canal and creates an acidic coat which aids in prevention of infection of
external auditory canal (Campos et al., 1998)

Cerumen is composed of lipids proteins, amino acids, carbohydrates, hormones, anti-bodies,

enzymes. Its products make it a reflection of physiological function of the body and a potential
antimicrobial agent. (Yassin et al., 1996). The acidity property it possesses also captures the
researcher’s attention on its anti-microbial property. Lack of ear wax may cause infection as it
serves antimicrobial property by physiological protection of ear canal, creating a low pH and
harsh atmosphere for pathogen and antimicrobial substance such as lysozyme which acts against
various bacteria (Bojrab et al 1996). Its absence leads to ear canal susceptible infections. This
therefore implies that ear wax has got some great antimicrobial activity

Although mortality from infectious disease has declined globally, bacterial infections still impact
the world’s most vulnerable populations. Bacterial infections including pneumonia and diarrhea
are major killers in young children. Tuberculosis and viral hepatitis are among the leading causes
of deaths due to infectious disease in adults living in low- and middle-income countries
(Singleton, 1999). Treatment for these devastating diseases exists but their use and effectiveness
face numerous challenges including timely diagnosis, drug resistance and access to healthcare.

Antibacterial property entails the ability of a certain compound to act against a certain bacterium.
It may involve inhibiting of viability or growth or germination of certain bacteria, for our case
we are testing the antibacterial property of human ear wax against pathogenic bacteria existing
within Juja town. Pathogenic bacteria strains are harmful and have been associated with the
release of toxins causing food poisoning and other infections like diarrhoea kidney failure, in
susceptible individuals, respiratory illness, and pneumonia, typhoid, gut infections (WHO,2002;
Kumar et al.,2007).

1
1.2 Problem statement

Problems associated with infection by pathogenic bacteria within Juja town has been of great
concern. Pathogenic bacteria which have been studied to cause illness in human including food
poisoning, urinary tract infections, respiratory illness, pneumonia, meningitis, haemolytic
uraemic syndrome in young children, diarrhoea. It is associated with symptoms like fever,
vomiting, abdominal pains, and anaemic conditions. Food poisoning, diarrhoea and gut related
infections has posed great challenge to the Juja community and Kiambu county at large. (The
main sources of the pathogenic bacteria have been observed to be food and water.). In
susceptible individuals, pathogenic bacteria have been observed to cause kidney failures thus
these bacteria are more life threatening in infants and people with weakened immune systems.
(Todar, 2007; Lim et al., 2010). In addition, antibiotic resistance has also become a big threat to
global health, it has risen in all parts of the world making it hard to treat infectious diseases.
(Parovic and Schutz, 2016.). Many cases of antibiotic resistance have also been experienced
within Juja area and Nairobi regions according to WHO, 2015 article and approximately 700,000
lives worldwide are lost annually due to disease resistance (Neill, 2016).

1.3 Justification
Due to the fact that absence of ear wax in the human ear has resulted to various ear microbial
infection such as outer ear canal, otitis external ear itching and some other properties ear wax
possesses i.e. acidic nature, presence of antibodies, hormones, lipids, enzymes (lysozyme). Ear
wax is thus considered to have great antibacterial property. It prevents entry of any microbial
agent into the ear, it has a lubricating and cleansing property, and it repels water, traps that
prevent entry of any insect of any microbial agent. This antibacterial property of ear wax could
help come up with a long-term solution against bacterial infections and resistance and especially
those caused by pathogenic bacteria strains. (Schwab et al.,2011).

The ability of ear wax to inhibit microbial growth or viability of bacterial growth at different
concentrations could be the first step for the remedy against infections caused by the pathogenic
bacteria, further tests could be carried out and included in vaccine production and antibiotics that
will act against them. This will help save infant deaths since the pathogenic bacteria is more life
threatening to infants. (Todar, 2007).

2
 Antibiotic resistance has been experienced in treatment of some pathogenic bacteria stains
(WHO, 2015; Neill, 2016), therefore addition of some active components maybe the ear wax
may help solve the menace and maintain the various drugs pharmacodynamics. Determination
of inhibition zones will help to show the various extents of antibacterial property.

1.4 Research questions

Does ear wax have antimicrobial property against pathogenic bacteria isolated within Juja town
at various concentrations?

Does ear wax have a greater or lower antibacterial activity than ciprofloxacin against pathogenic
bacteria isolated within Juja Town?

What are the common pathogenic bacteria found within food samples in Juja?

1.5 Hypothesis
HO: Ear wax lacks antibacterial properties against pathogenic bacteria isolated within Juja town.

H1: Ear wax has antibacterial properties against pathogenic bacteria isolated in Juja town.

1.6 Study objectives

1.6.1 General objectives
To determine antibacterial property of ear wax against pathogenic bacteria isolated within Juja
town
1.6.2 Specific objectives
- To evaluate antibacterial effect of earwax against pathogenic bacteria at various concentrations.

- To determine and compare the antibacterial activity of ear wax and ciprofloxacin against
pathogenic bacteria at different concentrations.

-To identify the common pathogenic bacteria found in food samples within Juja town.

3
 CHAPTER TWO.
2.0 LITERATURE REVIEW
2.1 Ear wax/ cerumen
Cerumen is formed as a result of normal physiological process. Ear wax is formed by sebaceous
glands, ceruminous glands and apocrine glands present at the outer one third of the human
external auditory canal and creates an acidic coat which aids in prevention of infection of
external auditory canal. Lack of ear wax may cause infection as it serves antimicrobial property
by physiological protection of ear canal creating a low pH and harsh environment for pathogens
and creating antimicrobial substance such as lysozyme, its absence leads to susceptible
infections. Sebaceous glands secrete sebum which is mostly a combination of fatty acids
modified apocrine sweat glands secretion that combines with sebum to form cerumen. Ear wax is
made of cerumen which is a yellowish-brown waxy substance. Ear wax protects the tissues of the
ear and helps to prevent infections by trapping irritants such as microorganisms dead skin cells,
sweat, oil, air, and dirt from atmosphere. Part of the ear canal is lined with fine hair called cilia
that trap particles that enter the ear and then move them towards the ear opening where wax can
be washed off. The action of the jaw movement during talking and chewing also serves to
remove the wax out of the canal. (Campos et al,.1998; Bojrab et al,.1996; Aung and
Mulley,2002; Lum et al.,2009; Yassin et al,.1996; Roland et al., 2008)

Ear wax is slightly acidic which discourages fungal growth in moist and dark environment of ear
canal. Without ear wax it would be almost impossible to avoid ear infections. Individuals vary in
how much wax their ears produce, some produce relatively large amounts while others produce
relatively smaller amounts. Factors like emotional stress, fear, pain, use of certain drugs, anxiety
tends to increase production of ear wax. Swimming has also been studied to show an increase in
production of ear wax. (Lum et al., 2009; Katauria A, and Katauria K 1967.)

2.2 Composition of cerumen

Cerumen is a mixture of keratinocyte from the outer part of external auditory canal and
secretions form sebaceous glands along with apocrine sweat glands. It contains 60% keratin, 6-
9% cholesterol, saturated and unsaturated long chained fatty acids, and alcohols 12-20%.
Glandular secretions coming from hair follicles of external auditory canal also mix with cerumen

4
and make it a sticky substance. Cerumen is present in 10% of all children and up to 57% of older
persons. (Lum et al., 2009; Roeser and Ballachanda 1997). It is also composed of lipids,
proteins, carbohydrates, amino acids, hormones, anti-bodies, immunoglobulin glycopeptides. It
also possesses some enzymes like lysozyme which is an antibacterial enzyme capable of
destroying bacterial cell walls. It’s composed of squalene whose concentration differs in gender
and age. Its acidic which discourages bacterial or fungal attack. It contains esters which have
long aliphatic four chains of carbon molecules which ensures there are insoluble in water.
(Yassin et al, 1996; Katauria A and Katauria K, 1967).

The common proteins present in the ear wax include antimicrobial peptides, e.g. hBD1-3, and
lactoferrin, LL37, BP1 and HNP1-3. These antibacterial peptides prevent bacteria and fungi
causing infections in the ear canal. (Schwaab et al, .2011). The human beta defensins (hBD1-3)
has a stronger antibacterial property on gram negative bacteria. The hBD2 has a strong
antibacterial property against E. coli, P aeruginosa an C. albicans. All hBD have proven to have
antibacterial effects. (Schneider et al.,2005; Harder et al.,2001). The human LL37 is a 37 amino
acid long C terminus with active antimicrobial component. It is expressed on leucocytes like
neutrophils, monocytes, epithelial cells like skin, respiratory tract, gastrointestinal tract. It has
antibacterial property against both gram negative and positive bacteria (Wang et al,2004;
Haussen et al.,2008). The Human lactoferrin is present in saliva, tears, milk, nasal mucosa,
granulocytes. It has proven to have antibacterial property against vibrio cholera, C. albicans and
Streptococcus mutants and E. coli (Stenfors et al.,2002; Schwaab et al 2011), The Human
bactericidal permeability increasing protein (BPI): - it is a single chain cationic protein divided
by proteolysis into two segments. It’s mainly seen in granules of neutrophils dermal fibro blasts.
It has a bacterial effect on gram negative bacteria (Reichel et al., 2003).

2.3 Functions of ear wax

The ear wax provides protection against bacteria fungi, insects, and water. It contains a sticky
wax that acts like a stopping point or traps bacteria, fungus. It helps in shedding dead tissues,
cleanse the ear to ensure vibrations can pass through easily to be transformed into sound (Bass et
al.,1997; Campos et al.,1998; Shapiro and Clarke, 2002). Lubrication is another function as it
prevents desiccation of skin within the ear canal, its lubricative property is due to its high lipid
content of sebum. Ear wax cleaning is done or removed during jaw movement especially during

5
chewing. It dislodges the unwanted particles attached to walls of ear canal increasing likelihood
of its removal (Bass et al.,1997; Saxby et al.,2013)

2.4 Antibacterial properties

Ear wax possess antibacterial enzyme (lysozyme) that is capable of destroying bacterial cell
walls. Its slightly acidic nature prevents thriving of bacteria. They possess antibodies that act
against various bacterial infections. Its trapping ability of microorganisms, sweat, dirt, dead skin
cells, oil, air which are always rich in microbes. Possession of common protein like human beta
defensins, antimicrobial peptides, lactoferrins which have shown a greater antibacterial property
against a number of bacteria both gram positive and gram-negative bacteria (Stone et al,.1984;
Schwaab et al.,2011; Hyslop, 1971,).

2.5 Forms of cerumen

The cerumen exists in two forms which include the wet type that is dominant and common
among Africans and European people (Overfield et al.,1985). It is moist and has a higher
concentration of lipid and pigment granules. It has 50% lipids and a smaller percentage of
cholesterol and squalene. It is honey brown, dark orange or dark brown. The other form is the
dry type- which is common among East Asians and Native Americans. It has only 20% lipid, low
moisture content and appears grey in colour (Yassin et al.,1996; Tomita et al.,2002; Bass et
al.,1997).

A specific gene determines whether people have wet or dry ear wax, the gene is referred to as
ATP binding cassette C11 gene. Dry type individual is homozygous for adenine while wet types
require at least one guanine. (Yoshiura et al.,2006; Guest et al.,2004; Diep,2014).

Figure 2.1 showing wet earwax Figure 2.2 showing dry earwax

6
(http:www.en.wikipedia.org).’Cerumen’

2.6 Bacteria.

Bacteria are prokaryotic microorganisms a few micrometres in length, their cell wall is made of
peptidoglycan and they exist in various shapes like sphere, rod and spirals. Main sources of
bacteria include soil water, food sources, air. they also have symbiotic and parasitic relationship
with plant and animals. (Woese et al., 1990). Bacteria may be harmful i.e. pathogenic or non-
pathogenic. All animal life on earth is dependent on bacteria for their survival and some possess
genes and enzymes important in synthesising vitamin B12, that is soluble in water and is
involved in metabolism of every cell in human body. It is a co factor in DNA synthesis fatty acid
amino acid metabolism and is also key in normal functioning of nervous system (Fang et al
2017; Moore and Warren 2012). Bacteria may also be important in nutrient cycle by recycling
nutrients such as nitrogen fixation in the environment. They may also provide nutrients needed to
sustain life by converting dissolved compound like methane to energy (Choi 2013; Glud et al
2013). In addition, the large amount of bacteria exists in gut flora and in the skin. Majority of
bacteria in the body are harmless due to the protective ability of immune system though many
are beneficial especially in the gut flora. (Sears, 2005)

Pathogenic bacteria are harmful and trigger an infection. Main sources of these bacterial include
air, water, soil. Example of these pathogenic bacteria from these sources include E.coli, coliform,
Vibrio cholerae, Salmonella typhi, Staphylococcus aureus, Mycobacteria tuberculosis,
Helicobacter pylori, Listeria monocytogens, Bacillus subtilis. These pathogenic bacteria cause
infectious diseases including; cholera, typhoid, diarrhoea, respiratory infection, gut infection,
pneumonia, tetanus (WHO, 2002; Kumar et al 2007).

Pathogenic bacteria can be treated with antibiotics classified as bacterialcidal; if they kill the
bacteria) or bacteriostatic; if they prevent bacterial growth. (Yonath and Bashan 2004; Cassels
2012).

2.7 Mechanism of action of pathogenic bacteria

Pathogenic bacteria act or cause damages directly or indirectly. They cause infection indirectly
as a result of inappropriate immune response triggered by an infection which may damage the
host cell. They act directly where by the pathogen attached to the host cell causes direct damage

7
as pathogen will use the host cell for nutrient and produce products, in addition they produce
toxins that cause must of direct damage e.g. endotoxins are release when bacteria lysis which is
after antibiotic treatment (Greenwood et al 2012; Nash 2015).

2.8 Bacteria isolation

Bacteria isolation entails sourcing of the bacteria for example water, soil, gut, food sources etc.,
its followed by transportation and preservation of samples, culturing, identification which entail
visualization of bacteria using microscope based on shape and staining properties. Identification
is done to determine microbe of interest (Cassel 2012; Beverage and Davies 1993).

CHAPTER THREE.

8
3.0 MATERIALS AND METHODS
3.1: Study area and sampling
The bacteria isolate will be obtained within Juja town, Kiambu county north of Nairobi the
capital city of Kenya. The isolates will be collected from meat samples, sardine and water
samples from various points in Juja town. Meat samples will be collected in sterile bags, water
sample collected in sterile bottle while sardine will be collected in sterile bags for transportation
to biochemistry lab in JKUAT for further preparation. .Ethical approval will be obtained and
earwax samples will be collected from 30 healthy individuals both male and female from within
the institution (JKUAT; College of Health Sciences), out of a population of 2400 students using
stratified systematic random sampling with our stratum group being two; male and female and
will use the following formula;

Total population= 2400

sample size=30

male population= 1300

female population=1100

General formula=stratum group/Total population*sample size

therefore; male sample size= male population/total population*sample size

female sample size= female population/total population*sample size

3.2 Study design and methods used.

3.2.1 Bacteria isolation,media preparation and culturing.
The various differential and selective media to be used will be prepared as per the
manufacturer’s instructions.
Meat will be blended, 1g of the sample weighed and introduced into 9ml of sterile water and
mixed properly. Serial dilution of up to 10 -5
will be done, from this dilution,1ml of aliquot will
be transferred onto freshly prepared various selective media, spread using sterile glass spreader
and incubated at 37 o c for 24 hrs. 10g of sardine samples will be weighed, introduced into 9ml
sterile water and mixed properly. Serial dilution of up to 10-5 will be done and 1ml of aliquot

9
transferred into freshly prepared various selective media using sterile glass spreader and
incubated at 37 o c for 24 hrs. About 1ml of untreated water sample will be plated in the various
selective growth media(Eosin methylene blue,Salmonella-shigella agar,mannitol salt
agar,MacConkey agar) and incubated at 37 o c for 24 hrs. After the growth of the bacteria, all the
bacteria isolate depending on the type will be cultured on nutrient agar using streaking method
for inhibition test.

3.2.2 Bacterial inhibition test using cerumen.

Approximately 1g of Cerumen obtained will be dissolved in sterile buffer containing 5% sodium
bicarbonate and 30% glycerol after which it will be serial diluted with saline buffer up to 10-2 and
used for bacterial inhibition test. Agar plate diffusion method will be used to introduce the
antibacterial agent where by, filter papers will be punched with a punching machine of about
6mm diameter. They will be then soaked in the prepared cerumen of different concentrations.
The soaked discs at desired concentrations will be placed on the agar surface on the different
plates as labelled according to their concentrations. The plates will be incubated for 7 days at 37
degrees Celsius. The diameter of zones of inhibition will be measured to determine minimum
inhibitory concentration. The antibacterial agent will be expected to diffuse into agar and inhibit
growth of the test microorganism for positive test.

3.3 Data analysis

Data obtained will be collected into excel worksheets and analyzed by ONE-WAY ANOVA.
Results will be presented in pictures and tables.

WORK PLAN
The research work will be carried out from September 2019 to April 2020. The breakdown is as
follows:

10
  2019

ACTIVITY SEPTEMB OCTOBER NOVEM DECEMBE JANUARY FEBRU MARC APRIL

ER BER R ARY H

Proposal writing                

Submission,,oral                
presentation of
the proposal

Sample                
collection

Progress report                

  2020

In vitro studies-                
start of the lab
work.

Finalising lab                
work

Project report                
compilation

Project report                
submission,
presentation

BUDGET.

The cost to be incurred for the project is as follows;

11
 ITEM TYPE OF ITEM QUANTITY COST(KSH)
NUMBER

1 Printing and binding services - 500

2 PETRIDISHES 2 dozen 2000

3 Internet 2500 megabytes 500

4 GLOVES 1BOX 500

5 STRILE EAR BUDS 1 PACKET 50

6 Duplication of reports - 200

7 Sample collection allowance - 500

8 Test tubes 2 dozen 1500

9 Sample collection bags - 150

10 GLYCEROL 100ML 200

11 SODIUMBICARBONATE 50g 200

12 Transport - 500

13 Stationery - 200

14 Ethical approval 1500/-

Total 8500/-

-Refrigerator, autoclave, weigh balance, biosafety cabinet, and microbial test organism will be
provided by the institution’s biochemistry laboratory.

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