KENYA COAST NATIONAL POLYTECHNIC

TRADE PROJECT

TITTLE: COMPERATAIVE STUDY OF

ANTIMICROBIAL EFFECT OF THREE COMMONLY
USED ANTIBIOTICS ON STAYPHYLOCOCCUS
AUREUS AND ECHERICHIA COLI

NAME: EDWARD NGURE

INDEX NO: 1061200

PAPER CODE: 2404

SUPERVISOR: MRS, MWAVITA

COURSE NAME: DIPLOMA IN APPLIED BIOLOGY

SERIES: JUNE/JULY 2021

TRADE PROJECT SUBMITTED TO THE KENYA

NATIONAL EXAMINATION COUNCIL IN PARTIAL
FULFIMENT FOR THE AWARD OF DIPLOMA IN
APPLIED BIOLOGY

i
 DECLARATION
THE KENYA NATIONAL EXAMINATION COUNCIL

DECLARATION FORM FOR DIPLOMA IN APPLIED BIOLOGY

NAME OF THE PROJECT: COMPARATIVE STUDY OF THE ANTIMICROBIAL EFFECT

OF THREE COMMONLY USED ANTIBIOTICS ON STAPHYLOCOCCUS AUREUS AND
ESCHERICHIA COLI.

CODE NUMBER OF THE PROJECT

THE CANDIDATE OF INDEX NUMBER

i)The project named above was approved by KNEC and supervised by

(SUP
ERVISOR NAME)

ii)I personally carried out the project whose report follows after this declaration

iii)I received no undue help from unauthorized persons other than the normal guidance from my
supervisor

iv)The report submitted to the council is the original work

SIGNATURE DATE

THE SUPERVISOR:

Declare that ,i supervised the above named candidates project and report contained herein is the
genuine work of the candidate

SIGNATURE OF THE SUPERVISOR DATE

NB:TO BE ATTACHED TO THE FIRST PAGE OF THE PROJECT

ii
 DEDICATION
This project is dedicated to the entire East African population especially Kenyans who
experience antibacterial disease more than once due to the resistance of microbial pathogens to
antibiotics. This will help to identify the right antibiotic to curb a certain disease to reduce
sequential suffering.

Last but not least I dedicate it to my loving grandmother who have seen me through this and my
mother for helping me.

iii
 ACKNOWLEDGEMENT
Much concern and appreciation to my grandmother, who have seen me through all this by
helping me get the right advice and acknowledgement to move on with my studies.

I also appreciate and give thanks to Mrs Mwavita who is my course coordinator who have guided
and supervised me on my project.

And lastly but not least I do acknowledge the presence of my invigilators who carried a
supervision on my presentations and showed me the way forward.

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Table of Contents
DECLARATION..............................................................................................................................................ii
DEDICATION................................................................................................................................................iii
ACKNOWLEDGEMENT.................................................................................................................................iv
ABSTRACT..................................................................................................................................................vii
CHAPTER ONE..............................................................................................................................................1
1.0 INTRODUCTION.....................................................................................................................................1
1.1 BACKGROUND INFORMATION...............................................................................................................1
1.2 STATEMENT OF THE PROBLEM..............................................................................................................2
1.3 PURPOSE OF THE STUDY........................................................................................................................2
1.4 SIGNIFICANCE OF THE STUDY...............................................................................................................2
1.5 RESEARCH OBJECTIVES..........................................................................................................................2
SAMPLING AND LIMITATIONS.....................................................................................................................2
CHAPTER TWO.............................................................................................................................................4
2.0 LITERATURE REVIEW..............................................................................................................................4
2.1 INRODUCTTION.....................................................................................................................................4
2.1.2 AMOXICILLIN......................................................................................................................................4
2.2.2 CEPHALEXIN........................................................................................................................................4
2.3 AZITHROMYCIN......................................................................................................................................5
2.4 AMOXILLIN,CEPHALEXIN,AZITHROMYCIN..............................................................................................5
CHAPTER THREE..........................................................................................................................................6
3.0 METHEDOLOGY.....................................................................................................................................6
3.1 INTRODUCTION.....................................................................................................................................6
3.2 IDENTIFACATION OF TEST ORGANISMS.................................................................................................7
3.3 ANTIBACTERIAL SENSITIVITY/SUSCEPTIBILITY TEST...............................................................................7
CHAPTER FOUR............................................................................................................................................9
4.1 RESULTS AND DATA ANALYSIS...............................................................................................................9
4.2 INTERPRETATION OF ZONES OF INHIBITION........................................................................................10
4.3 GRAPHS INDICATING INTERPRETATION OF ZONES OF INHIBITION BETWEEN STAPHYLOCOCCUS
AUREUS AND ESCHERICHIA COLI TO AMOXYCILLIN, CEPHALEXIN AND AZITHROMYCIN...........................12
4.4 CONCLUSION AND RECOMMENDATION.............................................................................................13
4.5 DISCUSSION.........................................................................................................................................13
4.6 CONCLUSSION.....................................................................................................................................14

v
4.7 RECOMMENDATIONS..........................................................................................................................15
REFERENCES..............................................................................................................................................16
APPENDICES..............................................................................................................................................17

vi
 ABSTRACT
Antibiotics are the drugs that either inhibit bacterial growth or kills the bacterial entirely .The
comparative analysis of the three most used antibiotics Amoxicillin, Cephalexin and
Azithromycin) was carried out using Staphylococcus aureus and Escherichia coli, as the test
organisms. Susceptibility test is used to determine the antimicrobial activity of an antibacterial
growth against an organism. many methods can be used to carry out this this test ;broth
dilution ,antimicrobial gradient .disc diffusion and automated instrument method.

Disc diffusion method was used to determine the antimicrobial strengths of the various
antibiotics at different concentrations of the test organisms.(Staphylococcus aureus and
Escherichia coli) The antibacterial activity of the antibiotics procured were tested in-vitro using
the antibiotics disc diffusion method.

The result showed that cephalexin had the highest antimicrobial effect on staphylococcus aureus
and Escherichia coli ,with zone of inhibition 32mm and 40mm respectively .Amoxicillin is also
the highly effective on staphylococcus aureus and a little bit effective on Escherichia coli with
zones of inhibition 30mm and 29mm respectively .Azithromycin was effective on
staphylococcus aureus with 22mm zone of inhibition and totally resistant to Escherichia coli with
no zone of inhibition .

Cephalexin is the best antibiotic amongst the three analyzed for the treatment of bacterial
infections implicating staphylococcus aureus and Escherichia coli, Amoxicillin is also effective
on staphylococcus aureus but not effective on Escherichia coli .From the research, it can be
recommended that whenever people are diagnosed of bacterial infections as a result of
staphylococcus aureus or Escherichia coli the medical personnel’s are advised to prescribe
Cephalexin as it has the highest antimicrobial effect on the organisms than the other antibiotics it
was compared with.

vii
 CHAPTER ONE

1.0 INTRODUCTION
An antibiotic also called an antibacterial is a type of antimicrobial drug used in the treatment and
preservation of bacterial infections. It may either kill or inhibit the growth of bacteria. A limited
number of antibiotics also posses antiprotozoal activity. Antibiotic are not effective against
viruses such as the common cold or influenza; drugs which inhibit viruses are termed as antiviral
drugs rather than antibiotics.

1.1 BACKGROUND INFORMATION

Antibiotics can be broad spectrum or narrow spectrum; Broad spectrum antibiotics are those
ones with activity against a wide range of gram positive and gram negative organisms while
narrow spectrum antibiotics has activity against one or few type of bacteria.

Antibiotics effectiveness and easy access has led to the over use prompting bacterial to develop
resistance. This has led to wide spread problems so much as to prompt the World Health
Organization to classify antimicrobial resistance as a serious threat that is no longer a prediction
for the future ,it is happening right now in every region of the world and has the potential to
affect anyone of any age in any country. Narrow spectrum antibiotics are those with activity
against one or few types of bacteria eg. Staphylococcus aureus and Escherichia coli. It is better
because once the infective agent is known as this limits the detrimental effects on the normal
bacterial flora.

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1.2 STATEMENT OF THE PROBLEM
With wide spread antibacterial resistance standard treatments has become in effective infections
persist may lead to spreading of infection hence becoming harder to treat a number of infections.

1.3 PURPOSE OF THE STUDY

The study is carried out to ascertain the antimicrobial effect of three commonly used antibiotics
on staphylococcus aureus and Escherichia coli and suggest the most appropriate antibiotic for
effective results on the microbes.

The research study is also part of fulfillment of diploma in applied biology.

1.4 SIGNIFICANCE OF THE STUDY

Due to rapid increase in mortality ,higher medical costs and longer hospital stays the study
ascertains the right antibiotic to inhibit or kill staphylococcus aureus and Escherichia coli.

1.5 RESEARCH OBJECTIVES

i)To determine the antimicrobial effects of amoxicillin on staphylococcus aureus and
Escherichia coli.

ii)To determine the antimicrobial effects of cephalexin on staphylococcus aureus and Escherichia
coli.

iii)To determine the antimicrobial effects of azithromycin on staphylococcus aureus and

Escherichia coli.

Iv)To compare the antimicrobial effects of Amoxicillin,Cephalexin,Azithromycin on

staphylococcus aureus and Escherichia coli.

SAMPLING AND LIMITATIONS

SAMPLING

Different pharmacies provide Amoxicillin, Cephalexin and Azithromycin for treatment of

bacteria. The sample for analysis was collected from three different locations .The sample for
analysis was collected in quantities of a tablet for each antibiotic.

2
LIMITATION

During the research study, various short comings were encountered which include the sampling
which were further apart and thus a lot of transport expenses was incurred.

3
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 INRODUCTTION

2.1.2 AMOXICILLIN
Staphylococcus aureus and Escherichia coli are zoonotic pathogen that cause various life
threatening diseases. Amoxicillin has been used to treat the pathogen for a long time but the
pathogens has evolved their resistance due to change in gene expression(Xinhua M (2015)
“Utilizing antibiotic agents effectively will preserve present day medication. Ghana News ep 15-
17.In the dosage time ,the appearance of resistant bacteria increase over time (NHS (2015)
National Health Scheme Antibiotics).The resistant bacteria are completely inhibited when the
cumulative percentage of time over a 24hour period that the drug concentration exceeds the
mutant prevention concentration(AHDEL(2011) American Heritage Dictionary of the English
language (5th edition).It provide reference for optimizing amoxicillin regimes to treat infections
caused by staphylococcus aureus.Amoxicillin works by preventing bacteria from forming cell
walls which kills the bacteria. Jones s .(2014) Antibiotics Simplified .Bartlett Publishers Pp 15-
17.

2.2.2 CEPHALEXIN
Cephalexin in an antibiotic that is effective against most gram-positive cocci particularly
Escherichia coli and Staphylococcus aureus mostly used to treat acute and chronic urinary tract
infections, upper and lower respiratory tract infections. It is administered orally as either 250mg
or 500mg capsules.

Cephalexin is a beta-lactam antibiotic. In a bacterial cell peptidoglycan gives the cell wall
mechanical stability. Cephalexin use a beta-lactam to inhibit the synthesis of peptidoglycan
forming bacterial cell wall.(WHO (2002) Prevention of Hospital Acquired Infection).

The beta-lactam binds to penicillin binding proteins,resultng in inhibition of the last phase of
peptidoglycan synthesis, which is a transpeptidation reaction required for bacterial peptidoglycan
cross-linking.

This activity results in the loss of cell viability and eventually leads to bacterial cell autolysis.

4
However the bacterial can obtain resistance to cephalexin by modifying the penicillin binding
protein, which alters the binding of cephalexin to their target site.

2.3 AZITHROMYCIN
It is an antibiotic widely used to treat chest infections such as pneumonia, nose infections such as
sinus, skin(lyme diseases) and some sexually transmitted infections. Mostly used in children to
treat ear infections.

Despite azithromycin being used in some countries to treat infections caused by gram-negative
pathogens no resistance breakpoint for Escherichia coli exists same as to staphylococcus aureus.
(copra, Hessse l, O’Neil A (2002) Discovery and Development New Anti-bacterial Drugs in
Pharmacochemistry Library )

2.4 AMOXILLIN,CEPHALEXIN,AZITHROMYCIN
Azithromycin works by binding to the bacterial and preventing the bacteria from producing
proteins that needs to survive while amoxicillin works by preventing bacteria from forming cell
walls, which kills the bacteria. Cephalexin on the other hand interferes with cell wall synthesis
by binding proteins inside the cellular wall leading to the destruction of the bacterial cell.

Azithromycin has the highest antimicrobial effect on staphylococcus aureus and Escherichia coli
among the three antibiotics.

5
 CHAPTER THREE

3.0 METHEDOLOGY

3.1 INTRODUCTION
This chapter involves the procedures and methods in terms of culturing and identification of test
organisms. Its also entails on preparation of nutrient agar.

PREPARATION OF NUTRIENT AGAR

REAGENTS USED

I. 28g of nutrient agar

II. Distilled water
III. Heat

EQUIPMENT USED

I. Petri dish
II. Autoclave
III. Wire loop
IV. Bursen burner

Nutrient agar contains nutrients that are suitable to subculture a wide range of
microorganisms and makes it an excellent agar media to check on purity before any
biochemical or serological test.

HOW TO PREPARE NUTRIENT AGAR

i)Suspend 28g of nutrient agar powder in 1l of distilled water.
ii)Mix and dissolve them completely
iii)Sterilize by autoclaving at 121C for 15 minutes.

6
 iv)Pour the liquid into the petri dish and wait for the medium to solidify. Be sure that
you are preparing the agar in the clean environment to prevent any contamination.
viii) Once the agar solidifies ,the agar is ready to use.

3.2 IDENTIFACATION OF TEST ORGANISMS

The test organisms were sub-cultured onto Nutrient agar plates and biochemical tests were
carried out include; Gram staining, indole test,catalase,test,coagulate test.

3.3 ANTIBACTERIAL SENSITIVITY/SUSCEPTIBILITY TEST

The minimum inhibitory (MIC) concentration was taken as the lowest concentration of different
antibiotics on the disc that would inhibit the growth of bacteria in Nutrient agar. The minimum
inhibitory concentration of the various antibiotics used are shown in the table below.

AMOXICILLIN (AM) 20ug

CEPHALEXIN (CPX) 5ug
AZITHROMYCIN (A) 15ug

The antibacterial activity of the antibiotics procured from the pharmacy were test in-vitro using
the Antibiotics Disc Diffusion Method. The antibiotic discs already have the antibiotics
incorporated in them at various potencies.

Nutrient agar plates were prepared and each plate was properly inoculated with the 24 hours
culture of the test organism in nutrient broth using sterile swab sticks with the Petridis lids in
place,they are kept for 3-5 minutes so that the surfaces of the agar dry.

Using a sterile forceps, the antibiotics discs were carefully placed on the inoculated plate
ensuring the discs were slightly pressed down to ensure adequate contact with agar.

Within 15-30 minutes of the antibiotic discs application, the plates were inverted and incubated
at 35C for 16-18 hours. After the incubation the plates were carefully examined for confluent or
near confluent growth and zones of inhibition.

Using a ruler, the diameter of the zones of inhibition were measured in millimeter (mm) from the
underside of the plate.

7
For confirmation the agar with diffusion method was carried out where a 2-field serial dilution of
the three antibiotics using sterile water as the diluent was done and it gave different
concentrations : 80%,60%,40% and 20%.Nutrient agar plates were prepared and each plate was
properly inoculated with each test organism using sterile swabs tics.

Wells were made using a sterile cork borer and well was carefully filled with different
concentrations of the antibiotics. The plates were incubated at 37C for 16-18 hours allowing the
antibiotics to diffuse properly into the nutrient agar.

After the incubation, the plates were observed for zones of inhibition. The zones of inhibition
were obtained by measuring the diameter using a ruler I millimeter(mm).The interpretation of
the zones of inhibition that is whether an organism is susceptible, intermediate or resistant to an
antibiotic was done based on CLSI (clinical laboratory standards institute).

ANTIMICROBIAL RESISTANT INTERMEDIATE SUSCEPTIBLE

AGENT

AMOXICILLIN < 19 - >20

CEPHALEXIN < 15 16-20 >21

AZITHROMYCIN < 13 14-22 >23

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 CHAPTER FOUR

4.1 RESULTS AND DATA ANALYSIS

The biochemical tests carried out on the organisms show that Staphylococcus aureus is coagulase
positive, catalase positive and gram positive cocci and Escherichia coli is coagulase
nagative,catalase negative, indole positive and a gram negative rod.

TEST GRAM STAIN INDOLE TEST CATALASE TEST COAGULASE

ORGANISMS TEST

1.Staphylococcus NA _ _ _
aureus

2.Escherichia coli _ _ _ _

KEY

NA-Not applicable

-= positive

-= negative

The sensitivity susceptibility test showed that staphylococcus aureus is susceptible to

AmoxIcillin,Cephalexin and intermediate to Azithromycin with the diameter of zones of
inhibition 30mm,32mm,22mm respectively while Escherichia coli is susceptible to cephalexin
with zone of inhibition 40mm resistant to Amoxycillin,with zone of inhibition 19mm and also
resistant Azithromycin with no zone of inhibition.

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4.2 INTERPRETATION OF ZONES OF INHIBITION

The sensitivity of staphylococcus aureus to amoxycillin,cephalexin, azithromycin.

ANTIBIOTICS POTENCY(ug) ZONE OF

INHIBITION(mm)

1 AMOXYCILLIN 20 30

2 CEPHALEXIN 5 32

3 AZITHROMYCIN 15 22

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 sensitivity of staphylococcus aureus

30

22

32

amxicillin cephalexin azitheromycin

The sensitivity of Escherichia coli to Amoxycillin,Cephalexin and Azithromycin

ANTIBIOTICS POTENCY ZONE OF INHIBITION

1 AMOXYCILLIN 20 19

2 CEPHALEXIN 5 40

3 AZITHROMYCIN 15 No number of zone of

inhibition

11
 sensitivity of escherichia coli

19

40

amoxycillin cephalexin azithromycin

Using the above, the organisms are reported as RESISTANT ‘intermediate/Moderately

susceptible.

Resistant ;A pathogen reported as ‘resistant’ implies that the infection it has caused will not
respond to treatment with the drug to which it is resistant irrespective of dose or site of infection.

Intermediate ;A pathogen reported as intermediately susceptible suggests that the infection it has
caused is likely to respond to treatment when the drug is used in larger doses than normal or
when the drug is concentrated at the site of infection e.g. in the urinary tract.

Susceptible; A pathogen reported as susceptible suggests that the infection it has caused is likely
to respond to treatment when the drug to which it is susceptible is used in normal recommended
doses and administered by an appropriate route.

The graphs below also show distribution of zone of inhibition on staphylococcus aureus and
Escherichia coli to amoxycillin,cephalexin and azithromycin.

4.3 GRAPHS INDICATING INTERPRETATION OF ZONES OF INHIBITION

BETWEEN STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI TO
AMOXYCILLIN, CEPHALEXIN AND AZITHROMYCIN
The sensitivity of staphylococcus aurous to amoxycillin,cephalexin and azithromycin

12
 45

40

35

30

25

20

15

10

0
cephalexin azithromycin amoxycillin

Series 1 Series 2 Series 3

The sensitivity of Escherichia coli to amoxycillin,cephalexin

and azithromycin

50
4.4
45
40
35
30
25
20
15
10
5
0
cephalexin azithromycin amoxycillin

Series 1 Series 2 Series 3

CONCLUSION AND RECOMMENDATION

4.5 DISCUSSION
This analysis was carried out to compare the antimicrobial strength of the three most common
antibiotics on some pathogenic microorganisms ,Staphylococcus aureus and Escherichia coli.
The antibacterial spectrum and efficiency against the test organisms were demonstrated.

13
From the sensitivity test with staphylococcus aureus ,cephalexin the highest zone of inhibition
followed by Amoxicillin and Azithromycin with diameters ,32mm,30mm,and 22mm
respectively .According to research the test organism ie staphylococcus aureus and Escherichia
coli is susceptible to cephalexin and amoxicillin and intermediate to azithromycin
staphylococcus aureus is most sensitive to cephalexin with Escherichia coli ,cephalexin has the
highest zone of inhibition followed by Amoxicillin with diameters,40mm and 22mm while
Azithromycin showed no zone of inhibition.

The test organism is susceptible to cephalexin and resistant to Amoxicillin and Azithromycin.
Escherichia coli most sensitive to cephalexin.

4.6 CONCLUSSION
It has been shown that the potency of antibiotics very depending on the test organism. From
analysis carried out, it can be concluded that cephalexin has the highest antimicrobial strength
against staphylococcus aureus and Escherichia coli .Amoxicillin is also effective on
staphylococcus aureus but not effective on Escherichia coli.

14
4.7 RECOMMENDATIONS
From the analysis above ,it can be recommended that whenever people are diagnosed of bacterial
infection as a result of staphylococcus aureus or Escherichia coli, the medical personnel’s are
advised to prescribe cephalexin as it has the highest antimicrobial effect on the organisms than
the other antibiotics it was compared with.

15
REFERENCES
1. AH DEL (2011) American Heritage Dictionary of the English Language (5th edition)

2. JONE S. (2014) Antibiotics Simplified. Bartlett Publishers Pp 15-17

3. NHS (2015) National Health Scheme Antibiotics.

4. WHO (2002) Prevention of Hospital Acquired Infection (2nd Edition)

5. WILEY J (2012) Chemical Analysis of Antibiotic Residues in Food (2 nd edition). Inc

Publications Pp 1-60

6. Xinhua M (2015) ‘Utilizing antibiotics agents effectively will preserve present day medication
.Ghana News Pp 15-17.S

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APPENDICES

17
18