ANTIBIOTICS SUSCEPTIBILITY OF Staphylococcus aureus: A COMPARATIVE STUDY

ANTIBIOTICS SUSCEPTIBILITY OF Staphylococcus aureus:
A COMPARATIVE STUDY

1. ANTIBIOTICS SUSCEPTIBILITY OF Staphylococcus aureus:
A COMPARATIVE STUDY
BY
ABUE SUNDAY YIOKI
20/MCB/133
SUBMITTED TO
DEPARTMENT OF MICROBIOLOGY
FACULTY OF BIOLOGYICAL SCIENCES
UNIVERSITY OF CROSS RIVER STATE
CALABAR.
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE IN
MICROBIOLOGY
SEPTEMBER, 2025
1

2. CERTIFICATION
This is to certify that this research work titled “ANTIBIOTICS SUSCEPTIBILITY
OF Staphylococcus Aureus: A COMPARATIVE STUDY” was carried out by ABUE
SUNDAY YIOKI with Registration No: 20/MCB/0133 of the Department of
Microbiology, Faculty of Biological Sciences University of Cross River State, under
the supervision of Dr. Tarh Jacqueline E.
ABUE SUNDAY YIOKI Signature:…………… Date:…………………
Student
DR. TARH JACQUELINE E .Signature:……………… Date:…………………..
Supervisor


2

3. DEDICATION
This research work is dedicated to the Almighty God for His Divine love and
providence.
3

4. ACKNOWLEDGEMENTS
First and foremost, I give all glory and gratitude to Almighty God for His
guidance, protection, and wisdom which have enabled me, Abue Sunday Yioki (Reg.
No. 20/MCB/133), to successfully complete this project.
I also wish to extend my heartfelt appreciation to my supervisor, Dr. (Mrs.)
Tarh Jacqueline Ebob, for her invaluable guidance, constructive criticism, and
constant encouragement throughout the course of this work. Her dedication and
support have been instrumental to the success of this project.
My sincere appreciation also goes to Comrade Ajaba Simon, Romanus Abue,
Julius Abue, Emmanuel Abue for their support, encouragement, and contributions
toward the completion of this project.
Finally, I am deeply grateful to my parents, Mrs. Abue Rose Ade and Mr. Abue
Augustine Ayebe, for their unwavering love, prayers, and sacrifices. Their support and
encouragement have been the foundation of my academic journey, and I remain
profoundly indebted to them.
4

5. ABSTRACT
The provision of safe drinking water remains a critical global challenge, particularly in rural
communities that depend on groundwater sources. Staphylococcus aureus is a major human
pathogen responsible for a wide range of infections, from gastrointestinal to life-threatening
systemic diseases. Its ability to develop resistance to multiple antibiotics has made it a
significant public health concern globally. This study was designed to determine and compare
the antibiotic susceptibility patterns of S. aureus isolates from house hold well water sources
in the University of Cross River State Staff quarters. Standard microbiological methods were
used to evaluate twelve well water samples and the antibiotic susceptibility testing performed
using disc diffusion methods against six commercially available antibiotics: penicillin G,
trimethoprim/sulphonamide, vancomycin, tetracycline, streptomycin, and chloramphenicol.
The results showed that seven 7(58.33%) of the well water samples were contaminated with
Staphylococcus spp., with 5(71%) of isolates being coagulase-positive. Antibiotic resistance
analysis revealed that 3 (42.86%) of isolates were resistant to penicillin G, 2 (28.57%) to
tetracycline, and 1 (14.29%) each to vancomycin and streptomycin. No resistance was
observed against trimethoprim/sulphonamide and chloramphenicol. Remarkable variations in
in susceptibility of the bacteria to the tested antibiotics was observed, showing this may
become a critical challenge in that area if an outbreak of infection with these bacteria occurs.
These findings indicate that wells used for house hold activities in the Cross River University
staff quarters, harbor antibiotic-resistant Staphylococci Thus, routine surveillance of
antimicrobial resistance patterns be intensified and that antibiotic stewardship programs be
strengthened to guide empirical therapy and reduce the misuse of antibiotics.
5

6. TABLE OF CONTENTS
TITLE PAGE
CERTIFICATION PAGE - - - - - - - I
DEDICATION - - - - - - - - - II
ACKNOWLEDGEMENTS - - - - - - - III
ABSTRACT- - - - - - - - - - IV
TABLE OF CONTENTS - - - - - - - - V
LIST OF TABLES- - - - - - - - - VI
CHAPTER ONE: INTRODUCTION
1.1 Background of the Study- - - - - - - 1
1.2 Aim and Objectives of the Study- - - - - - 2
CHAPTER TWO: LITERATURE REVIEW
2.1 Overview of Antimicrobial Agents- - - - - 4
2.2 Antibiotic Resistance in Staphylococcus aureus- - - 5
2.3 Mechanisms of Resistance in S. aureus- - - - - 7
2.4 Morphology and Characteristics of Staphylococcus aureus- - 8
2.4.1 Pathogenicity and Clinical Importance- - - - - 8
2.4.2 Methicillin-Resistant Staphylococcus aureus (MRSA)- - - 8
2.5 Global and Local Perspectives on Antibiotic Susceptibility Studies- 9
2.6 Empirical Review of Previous Studies- - - - - 9
CHAPTER THREE: MATERIALS AND METHODS
3.1 Research Design- - - - - - - - 10
3.2 Study Area- - - - - - - - - 10
3.3 Materials used - - - - - - - - 10
3.4Sample Collection and Confirmation - - - - - 10
3.5 Microscopy - - - - - - - - 10
3.5.1 Gram Stain- - - - - - - - - 10
3.5.2Motility Test - - - - - - - - - 11
3.6Biochemical Test for Confirmation - - - - - - 11
3.6.1 Catalase Test- - - - - - - - - 11
3.6.2 Oxidase Test- - - - - - - - - 11
6

7. 3.6.3Indole Test - - - - - - - - - 12
3.6.4Methyl-Red Test- - - - - - - - 12
3.6.5Citrate Test- - - - - - - - - 12
3.6.6Glucose Fermentation - - - - - - - 13
3.6.7Triple Sugar Agar (TSI) Test - - - - - - - 13
3.7Preparation of McFarland standard stored at room temperature (25
0
C) - 13
when not in use.- - - - - - - - 13
3.8Preparation and Standardization of the Inoculum - - - - 13
3.9Susceptibility test- - - - - - - - 13
CHAPTER FOUR: RESULTS
4.1: Morphology, cell count and physicochemical characteristics of isolates- 14
4.2 Percentage susceptibility/ resistance of Staphylococcus spp
isolates to the antibiotics- - - - - - - 14
CHAPTER FIVE:DISCUSSION, CONCLUSION, AND
RECOMMENDATIONS
5.2 Conclusion - - - - - - - - - 18
5.3 Recommendations - - - - - - - - 19
REFERENCES
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8. CHAPTER ONE
INTRODUCTION
1.1 Background of the Study
Staphylococcus aureus is a Gram-positive bacterium that has been recognized
as one of the most significant human pathogens due to its ability to cause a wide
spectrum of diseases ranging from minor skin infections to life-threatening conditions
such as sepsis, pneumonia, osteomyelitis, and endocarditis. It is also a common
colonizer of the human skin and mucosal surfaces, with the anterior nares being the
primary ecological niche (Ayepola et al., 2015). Colonization not only serves as a
reservoir for person-to-person transmission but also predisposes carriers to
endogenous infections.
Over the past decades, the treatment of S. aureus infections has become
increasingly complicated due to the widespread emergence of antimicrobial resistance.
The appearance of methicillin-resistant Staphylococcus aureus (MRSA) has posed a
significant public health challenge worldwide (Kaur & Chate, 2015). MRSA strains
are resistant to nearly all β-lactam antibiotics and often exhibit resistance to multiple
other antimicrobial classes, thus limiting therapeutic options. This resistance
contributes to prolonged hospital stays, increased healthcare costs, and higher
morbidity and mortality rates.
Recent studies have highlighted the variability in antibiotic susceptibility
profiles of S. aureus across different geographical regions and clinical settings. For
instance, while some isolates remain susceptible to glycopeptides such as vancomycin,
emerging resistance has been documented in several countries, raising concerns about
8

9. the sustainability of these last-line agents (McCarthy et al., 2015). Moreover,
community-associated MRSA strains, which tend to affect individuals without prior
healthcare exposure, have been increasingly reported among children and young
adults, complicating control measures (Alaklobi et al., 2015).
The persistence of S. aureus infections is not only driven by antimicrobial
resistance but also by its virulence factors, including the ability to form biofilms on
medical devices and host tissues. Biofilm formation enhances resistance to host
defenses and antibiotics, making infections more difficult to eradicate (McCarthy et
al., 2015).
Given these challenges, continuous surveillance of S. aureus antibiotic
susceptibility patterns is essential to guide effective empirical therapy and infection
control policies. Comparative studies assessing susceptibility profiles of clinical and
carriage isolates provide critical insights for antimicrobial stewardship programs and
public health interventions. This study therefore focuses on the comparative antibiotic
susceptibility of S. aureus to inform treatment strategies and strengthen control
measures in the study area.
1.2 Aim and Objectives of the Study
Aim
The primary aim of this study is to determine and compare the antibiotic susceptibility
patterns of Staphylococcus aureus isolates obtained from different sources, in order to
generate evidence that will guide effective treatment, antimicrobial stewardship, and
infection control strategies.
Specific Objectives
9

10. The study seeks to:
i. Isolate and identify Staphylococcus aureus from clinical samples and carriage
sources within the study area.
ii. Determine the prevalence of methicillin-resistant Staphylococcus aureus
(MRSA) among the isolates.
iii. Evaluate the antibiotic susceptibility profiles of S. aureus isolates against
commonly used antimicrobial agents.
10

11. CHAPTER TWO
LITERATURE REVIEW
2.1 Overview of Antimicrobial Agents
Antimicrobial agents are chemical substances that inhibit the growth of, or
destroy, microorganisms such as bacteria, fungi, and viruses. They are classified based
on their mechanism of action, chemical structure, or spectrum of activity. Since their
introduction in the early 20th century, antimicrobial agents have revolutionized
medicine, reducing morbidity and mortality associated with infectious diseases
(Ventola, 2015).
Antimicrobial agents are substances that inhibit the growth of or destroy
microorganisms, and they form the foundation of modern infection management. They
are generally classified into major groups based on their mechanism of action:
inhibitors of cell wall synthesis (e.g., β-lactams), inhibitors of protein synthesis (e.g.,
macrolides, aminoglycosides, tetracyclines), inhibitors of nucleic acid synthesis (e.g.,
fluoroquinolones), and metabolic antagonists (e.g., sulfonamides) (Kaur & Chate,
2015). Despite the clinical benefits of these agents, their widespread misuse and
overuse in both humans and livestock have accelerated the emergence of resistant
strains, thereby undermining their effectiveness. The World Health Organization
(WHO) identified antimicrobial resistance as one of the most critical global health
threats, with Staphylococcus aureus being among the priority pathogens (WHO,
2015).
Broadly, antimicrobials can be categorized into antibacterials, antifungals,
antivirals, and antiparasitics, with antibiotics forming the most widely used group.
11

12. Antibiotics act through various mechanisms, including inhibition of cell wall synthesis
(e.g., β-lactams such as penicillins and cephalosporins), inhibition of protein synthesis
(e.g., aminoglycosides and tetracyclines), inhibition of nucleic acid synthesis (e.g.,
fluoroquinolones), and disruption of metabolic pathways (e.g., sulfonamides) (Fair &
Tor, 2014).
The effectiveness of antimicrobial agents is influenced by factors such as the
type of microorganism, site of infection, pharmacokinetics of the drug, and host
immunity. However, widespread and often indiscriminate use of antibiotics has led to
the emergence of antimicrobial resistance (AMR), which is a growing global health
crisis (World Health Organization [WHO], 2019). Resistant pathogens such as
Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli have
significantly reduced the efficacy of first-line antibiotics, leading to prolonged illness,
higher healthcare costs, and increased mortality (Prestinaci, Pezzotti, & Pantosti,
2015).
The World Health Organization has repeatedly emphasized the urgent need for
rational use of antimicrobial agents, antimicrobial stewardship programs, and
continuous monitoring of resistance patterns to preserve the effectiveness of existing
drugs (WHO, 2019). Thus, understanding antimicrobial agents and their resistance
patterns is essential in guiding effective therapeutic choices and in the development of
new treatment strategies.
2.2 Antibiotic Resistance in Staphylococcus aureus
Staphylococcus aureus is the most virulent and pathogenic gram positive
bacteria of human. S. aureus is a versatile bacteria naturally equipped with a variety of
12

13. virulence factors that contribute to its pathogenicity. Therefore, S. aureus causes a
wide variety of infections, ranging from simple superficial skin lesions like folliculitis
to deep-seated abscesses and various pyogenic infections like endocarditis,
osteomyelitis, pneumonia, septicemia, and meningitis. Furthermore, it is also
responsible for a number of toxin-mediated illnesses, including food poisoning, toxic
shock syndrome, and staphylococcal scalded skin syndrome. However, S.
aureus exists as normal flora on various body parts such as nasal passages, throat,
intestines, skin, and mucous membranes in approximately 30% of healthy people
(Murray et al., 2021).
Staphylococcus aureus usually have unique propensity of developing resistance
to commonly used antibiotics. It’s common antimicrobial resistance mechanisms
includes antibiotic inactivation by enzymes, lower affinity for antibiotics due to
altered targets, efflux pumps, and antibiotic trapping. Methicillin resistant
Staphylococcus aureus (MRSA) is a strain of Staphylococcus aureus that is resistant
to all β-lactam antibiotics, including methicillin, oxacillin, amoxicillin, and penicillin,
as well as other β-lactams that are commonly used to treat staphylococcal infections
(Perera and Hay 2015).
S. aureus has developed resistance to multiple classes of antibiotics since the
introduction of penicillin in the 1940s. Penicillin resistance was first reported within a
few years of its clinical use, followed by resistance to methicillin in the early 1960s,
which gave rise to methicillin-resistant Staphylococcus aureus (MRSA) (McCarthy et
al., 2015). Today, MRSA remains a leading cause of healthcare-associated and
community-acquired infections worldwide. Resistance in S. aureus is often
13

14. multifactorial, including resistance to macrolides, aminoglycosides, fluoroquinolones,
and even reduced susceptibility to glycopeptides such as vancomycin (Ayepola et al.,
2015).
14

15. 2.3 Mechanisms of Resistance in S. aureus
Resistance mechanisms in S. aureus are diverse. Methicillin resistance is
mediated by the mecA gene, which encodes an altered penicillin-binding protein
(PBP2a) with reduced affinity for β-lactam antibiotics (Kaur & Chate, 2015).
Resistance to macrolides occurs via efflux pumps or ribosomal target site
modification, while aminoglycoside resistance is conferred through enzymatic
inactivation. Fluoroquinolone resistance is associated with mutations in DNA gyrase
(gyrA) and topoisomerase IV (parC) genes. Importantly, biofilm formation enhances
tolerance to antibiotics and protects S. aureus from host immune responses, making
infections persistent and recurrent (McCarthy et al., 2015).
In both hospital and community settings, there have been reports of significant
changes in S. aureus susceptibility to beta-lactam antibiotics, particularly penicillin
and cephalosporin. In some countries, like Nepal and South west Nigeria MRSA
makes up to 75% of all S. aureus isolates in hospitals. Multidrug-resistant strains limit
the therapeutic options, creating an economic and social burden to the healthcare
system. Horizontal gene transfer in the hospital setting is responsible for
disseminating antibiotic resistant determinants. Chromosomal mutation antibiotics
selection is also responsible for antibiotics resistance. In Ethiopia, antibiotics can be
obtained over-the-counter without prescription. This type of behavior along with
others of a similar practices mentioned before contributes to the emergence and spread
of antimicrobial resistance, rendering the most infections non-treatable. 
15

16. 2.4 Morphology and Characteristics of Staphylococcus aureus
S. aureus is a Gram-positive coccus that appears in grape-like clusters under
the microscope. It is catalase- and coagulase-positive, non-motile, and facultatively
anaerobic. Colonies are typically golden-yellow on nutrient agar, which is the origin
of its species name "aureus." The organism is widely distributed in the environment
and colonizes up to 30% of the healthy human population, with the anterior nares
being the most common reservoir (Ayepola et al., 2015).
2.4.1 Pathogenicity and Clinical Importance
The pathogenicity of S. aureus is attributed to a variety of virulence factors, including
surface proteins (adhesins), toxins (such as Panton-Valentine leukocidin), enzymes
(coagulase, hyaluronidase), and biofilm formation. Clinically, S. aureus causes skin
and soft tissue infections, pneumonia, bloodstream infections, osteomyelitis, and
endocarditis. Severe invasive infections are often associated with high morbidity and
mortality rates (McCarthy et al., 2015).
2.4.2 Methicillin-Resistant Staphylococcus aureus (MRSA)
MRSA is one of the most significant multidrug-resistant pathogens globally. It is
divided into healthcare-associated MRSA (HA-MRSA) and community-associated
MRSA (CA-MRSA), which differ in epidemiology, genetic makeup, and resistance
patterns. CA-MRSA often carries the staphylococcal cassette chromosome mec
(SCCmec) type IV and produces toxins such as Panton-Valentine leukocidin, while
HA-MRSA generally carries SCCmec types I, II, or III (Alaklobi et al., 2015). Both
forms pose treatment challenges due to their multidrug resistance profiles and
potential for rapid transmission.
16

17. 2.5 Global and Local Perspectives on Antibiotic Susceptibility Studies
Globally, studies have shown that MRSA prevalence varies across regions. In Europe
and North America, MRSA rates are declining due to effective infection-control
practices, while in Africa and Asia, the prevalence remains high due to limited
resources, unregulated antibiotic use, and weak surveillance systems (Kaur & Chate,
2015). Locally, studies in Nigeria and other African countries have demonstrated a
high prevalence of multidrug-resistant S. aureus strains, underscoring the need for
routine surveillance to inform empirical therapy (Ayepola et al., 2015).
2.6 Empirical Review of Previous Studies
Several studies have investigated the antimicrobial susceptibility of S. aureus.
Ayepola et al. (2015) conducted a molecular and antimicrobial susceptibility study in
Southwest Nigeria and reported high resistance rates to penicillin, erythromycin, and
tetracycline, while most isolates remained susceptible to vancomycin and linezolid.
Kaur and Chate (2015) highlighted the emergence of reduced susceptibility to newer
agents such as linezolid and tigecycline in some MRSA strains in India. Alaklobi et al.
(2015) reported a significant prevalence of CA-MRSA among children in Riyadh,
Saudi Arabia, with resistance to multiple classes of antibiotics. McCarthy et al. (2015)
emphasized the role of biofilms in enhancing resistance and persistence of MRSA in
both healthcare and community settings. Collectively, these studies reinforce the
urgent need for comparative susceptibility studies across regions to guide treatment
policies and antimicrobial stewardship programs.
17

18. CHAPTER THREE
MATERIALS AND METHODS
3.1 Research Design
This study adopted an experimental laboratory-based design, focusing on the
isolation, identification, and comparative antibiotic susceptibility testing of Staphylococcus
aureus isolates. The design was appropriate because it enabled the controlled investigation of
susceptibility patterns to different antibiotics using standardized microbiological techniques.
3.2 Study Area
The study was conducted in Microbiology Department, UNICROSS, Calabar.
Samples were collected from both clinical facilities and community settings to provide a
comparative analysis between infection-derived and carriage-derived isolates.
3.3 Materials used
The following materials were used; plastic petri dish, wire-loop, macro-pipette,
pressure pot, incubator, weighing balance, test-tube, proteus bacteria, Nutrient agar, test-tube
holder, Macfarland standard, Normal saline, plastic ruler, cornical flask, foil paper, masking
tape, marker, antibiotic disk, sterile forceps and spatula.
3.4Sample Collection and Confirmation
Staphylococus spp stock cultures were collected from University of Calabar Cross River State
Research Laboratory. These samples were collected with the consent of those involved, and
immediately taken to the microbiology laboratory for proper analysis. Upon arrival at the
laboratory, the samples were inoculated on Nutrient Agar (NA) and Mannitol salt agar plates
using streaking method. They were incubated at 37
0
C aerobically for 24 hours and examined
for growth. The samples were identified and characterized by standard method based on the
colony morphology, colony pigment, Gram stain and biochemical tests.
3.5 Microscopy
3.5.1 Gram Stain
This technique helps to identify pathogens in specimens and cultures by their Gram reactions
which are reported as Gram negative or Gram positive as well as their cell morphology.
A smear was prepared on a slide by transferring the inoculum to be examined into a drop of
suspension medium (distilled water) using an inoculation loop, spread evenly and allowed to
air dry. This was flooded with crystal violet stain (enough to cover the specimen) and allowed
for 30 seconds, and was gently rinsed with distilled water and covered with few drops of
iodine solution for 30 seconds, then it was rinsed with distilled water. The decolorizer
18

19. (acetone or ethanol) was poured on the slide until enough of the color and drip off clear. Then
immediately, the slide was rinsed with distilled water after 5 seconds. The basic counter stain
(safranin) was used to flood the slide and allowed for 20 seconds, then washed off with
distilled water. The stained swear slide was examined and the microscope using the oil
immersion objective (X100) to observe the cell morphology.
3.5.2Motility Test
This test is done to determine motile and non0-motile organisms. organisms possessing
locomotive organelles such as cilia, flagella or pseudopodia. No special reagent was used to
perform the experiment and it was determined by using Transmitted Light Microscope.
A small drop of suspension was applied on a slide and covered with a cover slip. The slides
were sealed with nail varnish to prevent drying out. The preparation was then examined
microscopically for motile organisms, using the 10x and 40 objectives and result was
recorded.
3.6Biochemical Test for Confirmation
3.6.1 Catalase Test
This test is used to differentiate those bacteria that produce the enzyme catalase such as
Staphylococci, from non-catalase bacteria such as Streptococcus (Cheesbrough, 2010).
About 2-3 ml of 3% hydrogen peroxide ((H2O2) solution was added to the test-tube and using
a sterile wooden stick applicator, a small amount of the test organism was collected from a
well isolated 18-24hours colony and placed into the test-tube against a dark background. The
tubes were observed for immediate bubble formation and result recorded.
3.6.2 Oxidase Test
This test assist in the identification of Vibrio, Aeromonas, Pseudomonas speciesetc, and
differentiates them from members of Enterobacteriaceae, due to their ability to produce the
enzyme cytochrome oxidase.
Watman filter paper was impregnated with drops of para-phynylenediaminehydrochloride
using a sterile syringe. An inoculum was picked using a sterile wooden applicator stick and
smeared on the drops of the reagents. The resultant colour change was observed within 5-10
seconds and result was recorded.
3.6.3Indole Test
About 1.5g of peptone agar was weighed into a conical flask and dissolved with 100ml of
distilled water. Aluminum foil was used to seal the flask and was then autoclaved at 121
0
C
for 15 minutes. This was allowed to cool at ambient temperature and about 5ml was
measured into each test-tube. The tryptophan broth was inoculated with the organisms under
19

20. investigation and incubated at 37
0
C for 24 hours and 5 drops of indole reagent was added into
each test tubes and shaken.The test tubes were allowed to stand undisturbed for 10 to 20
seconds and observed for a colour change at the surface layer and result was recorded.
Formation of pink to red color (“Cherry-red ring”) in the reagent layer on top of the medium
within seconds of adding the reagent indicate a positive test. Negative: No colour change
even after the addition of the appropriate reagent.
3.6.4Methyl-Red Test
About 1.5g of peptone agar was weighed into a conical flask and dissolved with 100ml of
distilled water. This was sealed with aluminum foil and autoclaved at 121
0
C for 15 minutes.
It was then allowed to cool for about 45
0
-50
0
and 5ml was measured into each test tube. The
tryptophan broth was then inoculated with the test organism and incubated at 37
0
C for 24
hours and 4 drops of methyl red reagent were added into each test tube. The test tubes were
shaken and observed for colour change and result was recorded.
3.6.5Citrate Test
About 4.6g of citrate agar was weighed into a conicalflask and dissolves in 200ml of distilled
water to yield 10 plates. it was then sealed and heated over a bursen flame to boil and
dissolve completely. It was then autoclaved at 121
0
C for 15 minutes and allowed to cool at
ambient temperature. It was then poured into the petri dishes and allowed to solidify and
inoculated with the organism under investigation. This was incubated at 37
0
c for 24 hours.
The plates were observed colour change and result recorded.
3.6.6Glucose Fermentation
About 6.3g of peptone was weighed into a conical flask and dissolved in 420ml of distilled
water. This was then sealed with aluminum foil and autoclaved at 121
0
C. The peptone broth
was allowed to cool. Approximately 3.1g of glucose-D (monohydrate) was measured into
10ml of the peptone water and the tubes were inoculated at 37
0
C for 24hours and observed
for acid and gas production and result was the recorded.
20

21. 3.6.7Triple Sugar Agar (TSI) Test
About 11.8g of triple sugar agar was weighed into a conical flask and dissolved with 200ml
of distilled water, measured into each test tube and autoclaved at 121
0
C for 15minutes. This
was allowed to cool for about 45-50
0
Cand slanted to solidify. The tubes were then
inoculatedand stabbed with the organism under investigation and incubated at 37
0
C for 24
hours. The tubes were observed for colour change, gas and hydrogen sulphide production.
3.7Preparation of McFarland standard
McFarland standard was prepared by using 1% solution of anhydrous Barium Chloride
(Bacl2) and 1% sulfuric acid solution (H2SO4). 0.5ml of 1% Barium chloride (BaCl2) with
9.95ml of 1% sulfuric acid (H2SO4) solution was mixed together in a test tube in their specific
proportion to form a turbid suspension. The mixture was placed in a foil covered screw-cap
tube. The standard was stored at room temperature (25
0
C) when not in use.
3.8Preparation and Standardization of the Inoculum
With a sterile wire loop, 3-5 isolate colonies of the test organism were picked from a bijou
bottled and emulsified in 4-5ml of sterile physiological saline to make a suspension of the test
organism. The test tubes were incubated for a few hours at 35
0
C until it becomes slightly
turbid and it was diluted serially to match a turbidity standard (McFarland standard already
prepared).
3.9Susceptibility test
Antibiotic susceptibility test was carried out on Mueller Hinton Agar following
recommendations of the national committee for clinical laboratory standards (2014).
Commercially prepared antibiotics discs were used, employing Kirby Bauer disc diffusion
method (Cheesbrough, 2010). A plate of Mueller Hinton agar was inoculated with the test
organism using pour plating method. About 1ml of the standardized organism was picked
from the test tube into a petri-dish aseptically and then the already prepared agar was poured
aseptically and mixed gently and allowed to solidify.
Using a sterile forceps, the appropriate antimicrobial disc was placed evenly distributed on
the inoculated plates and within 30 minutes of applying the discs, the plates were sealed,
inverted and incubated aerobically at 37
0
C for 24 hours. The resulting zones of inhibition or
clearance were measured in millimeters using a calibrated meter rule.
21

22. CHAPTER FOUR
RESULTS
4.1: Morphology, cell count and physicochemical characteristics of isolates
The occurrence and distribution of Staphylococcus spp. in well water samples in UNICROSS
quarters shows that out of the twelve wells sampled, seven 7(58.33%) yielded
Staphylococcus species with colony counts ranging from 1-4X10
10
for each positive sample.
From the Cultural and cellular morphology, microscopic examination revealed Gram-positive
cocci with 5(71.43%) occurring in clusters and some 2(28.57%) as tetrads. All presumptive
Staphylococcus isolates exhibited yellow, smooth, raised, and opaque colonial morphology
(Table 1). Also, the biochemical characteristics showed that two 2 (28.57%) were coagulase
negative (Table 1)
4.2 Percentage susceptibility/ resistance of Staphylococcus spp isolates to the antibiotics
Antibiotic resistance analysis revealed that 3 (42.86%) of isolates were resistant to penicillin
G, 2 (28.57%) to tetracycline, and 1 (14.29%) each to vancomycin and streptomycin. No
resistance was observed against trimethoprim/sulphonamide and chloramphenicol (Table 3).
22

23. Table 1: Morphology, cell count and physicochemical characteristics of isolates
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2 YellowSmoothRaisedOpaque1×10
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3 YellowSmoothRaisedOpaque1×10
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+CocciCluster/tetrad++-
5 YellowSmoothRaisedOpaque2×10
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+CocciCluster +++
6 YellowSmoothRaisedOpaque2×10
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+CocciCluster +++
7 YellowSmoothRaisedOpaque3×10
10
+CocciCluster +++
8 YellowSmoothRaisedOpaque4×10
10
+CocciCluster/tetrad+++
9 YellowSmoothRaisedOpaque4×10
10
+CocciCluster/tetrad++-
23

24. Table 2: Antimicrobial susceptibility profile of Staphylococcus species isolates.
PG=
Penicillin G, TS= Trimethoprim/Sulphonamide, V= Vancomycin, T= Tetracycline, S=
steptomycin C chloramphenicol, S sensitive, I intermediate and R=Resistant
24
IsolatePG TS V T S C
2 S S S S S S
3 R S R R R I
5 S S S S S S
6 S S S I S S
7 S I S I S S
8 R I S R I I
9 R I S I I I

25. Table 3: Percentage susceptibility/ resistance of Staphylococcus spp isolates to the antibiotics
Antibiotics % Susceptibility% Intermediate % Resistant
PG10 µg 4 (57.14) 0 3 (42.86)
TS25 µg 4 (57.14) 3 (42.86) 0
V30 µg 6 (85.71) 0 1 (14.29)
T30 µg 2 (28.57) 3 (42.86) 2 (28.57)
S30 µg 4(57.14) 2 (28.57) 1 (14.29)
C30µg 4 (57.14) 3 (42.86) 0
Key: PG= Penicillin G, TS= Trimethoprim/Sulphonamide, V= Vancomycin, T= Tetracycline,
S= steptomycin C chloramphenicol.
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26. CHAPTER FIVE
DISCUSSION, CONCLUSION, AND RECOMMENDATIONS
5.1Discussion
The present study revealed a concerning 58.33% contamination rate of well water samples
with Staphylococcus species in UNICROSS staff quarters. This finding aligns with previous
reports by Adesoji et al. (2019) in Dutsin-Ma community, Katsina State, and Osunla, (2025)
in in Selected Wells in Akungba-Akoko, indicating a global pattern of Staphylococcus
contamination in drinking water sources.
The high contamination rate likely reflects poor hygiene and sanitary conditions
around water sources (Osunla, 2025). The proximity of wells to waste dumps and latrines,
characteristic of the AkungbaAkoko community setting, represents a significant
contamination source (Osunla, 2025). This situation is particularly problematic given that
71% of isolated Staphylococcus species were coagulase-positive, indicating the
predominance of potentially pathogenic strains. The antibiotic resistance profile revealed
concerning patterns, with highest resistance to penicillin G 3(42.86%). This finding
corroborates previous studies by Adesoji et al. (2019 and Akinola, (2022), who reported
similar penicillin resistance in S. aureus from various sources. Similar resistance trends have
been reported in Nigeria and other African countries, where overuse and misuse of antibiotics
drive the emergence of multidrug-resistant S. aureus (Ayepola et al., 2015).
The observed β-lactam resistance likely results from β-lactamase production, an
enzyme that hydrolyzes β-lactam rings in these antibiotics. The presence of antibiotic-
resistant Staphylococcus species in drinking water poses significant public health
implications. Although S. aureus is not a traditional fecal contamination indicator, its
presence in domestic water supplies is concerning due to potential enterotoxin production and
food poisoning risks (Osunla, 2025). Moreover, the potential for horizontal gene transfer of
resistance determinants to gastrointestinal tract bacteria represents a significant concern for
antimicrobial therapy effectiveness. The resistance patterns observed may reflect broader
issues including antibiotic misuse, poor sanitation practices, and inadequate waste
management in the study area. The similarity in resistance patterns between clinical and
environmental isolates suggests possible epidemiological links, warranting further
investigation.
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27. 5.2 Conclusion
This study demonstrates significant Staphylococcus contamination in well water sources from
UNICROSS staff quarters, with concerning antibiotic resistance patterns. The findings
highlight the urgent need for improved water quality management and community health
interventions.
5.3 Recommendations
Government agencies should prioritize the provision of treated piped water and properly
constructed boreholes to reduce reliance on potentially contaminated wells.
Regular disinfection of existing water sources should be implemented and monitored by
appropriate government agencies responsible for water quality assurance.
Comprehensive community education programs should be established to promote proper
sanitation practices, appropriate sewage disposal methods and awareness of waterborne
disease risks.
Current antibiotic use policies require reassessment to address the growing problem of
antimicrobial resistance, coupled with research and development of new therapeutic agents.
Systematic microbiological monitoring of community water sources should be established to
ensure ongoing water safety and early detection of contamination events. Improved solid
waste management systems should be implemented to reduce environmental contamination
sources around water supplies
Public health education on rational antibiotic use should be intensified at community levels.
Further research should be conducted using larger sample sizes and molecular methods to
identify resistance genes.
27

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