KWARA STATE UNIVERSITY MALETE

The University for Community Development

FACULTY OF PURE AND APPLIED SCIENCES

ANTIBIOTIC RESISTANCE OF BACTERIA ISOLATES OBTAINED

FROM FEEDMILL

BY

ADEYEYE FOLASADE OLAMIDE

22D/57MB/2579

AUGUST, 2025.
 TOPIC ANTIBIOTIC RESISTANCE OF BACTERIA ISOLATES
OBTAINED FROM FEEDMILL

BY

ADEYEYE FOLASADE OLAMIDE

22D/57MB/2579

A Research Project Submitted to the Department of Microbiology,

Faculty of Pure and Applied Sciences, Kwara State University, Malete,
In Partial Fulfilment of the Requirements for the Award of Bachelor of
Science ([Link].) Degree of Microbiology.

AUGUST, 2025.

ii
 DECLARATION

I hereby declare that this research project titled “Antibiotic Resistance of Bacteria Isolates

Obtained from Feed mill” is my work and has not been submitted by any other person for

any degree or qualification at any higher institution. I also declare that the information

provided therein are mine and those that are not mine are properly acknowledged.

Adeyeye Folasade Olamide ______________

Name of student Signature and Date

iii
 CERTIFICATION

This is to certify that the research project titled “Antibiotic Resistance of Bacteria Isolates

Obtained from Feed mill” was carried out by ADEYEYE FOLASADE OLAMIDE

(22D/57MB/2579). The project was read and approved as meeting the requirements for the

award of Bachelor of Science ([Link].) in Microbiology, Faculty of Pure and Applied Sciences,

Kwara State University, Malete.

_______________________ ______________

Dr. W.T. Aborishade Date

(Project Supervisor)

_______________________ ______________

Ass. Prof. (Mrs.) A.E Ajiboye Date

(Head of Department)

_______________________ _____________

External Examiner Date

iv
 DEDICATION

This project is dedicated to Almighty God.

v
 ACKNOWLEDGEMENT

All thanks and adoration go to almighty God, the most esteem and the creator of all universe,

who has given me a grace to start this project in peace and end it in blessing. He has given me

the incentive towards my academy, also who made the completion of the task a great success.

My special thank goes to my effective supervisor Dr. Aborishade who has shown fatherly

love through his support, words of encouragement and advise, I pray you continue to grow in

wisdom. My profound gratitude also goes to the H.O.D, Dr. Mrs. A.E. Ajiboye, and other

lecturers for inspiring me to be a tourism practitioner, may God bless you all.

My profound gratitude and appreciation go to my parents, who has always been there for me.

I pray you eat the fruits of your labor. I say a very big thank you to my best friend,

Abdulmalik Olaitan Kasaba, also to my departmental best friends from way back in 100

level, Afolabi Bolaji, Lere Mujeeb, Amirat, Adam Yeseeroh, I say thank you for never giving

up on me. May we all meet at the top!

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 LIST OF TABLES

Table 1: Morphological Features of Bacteria Isolates Obtained from Feed mill.....................39

Table 2: Biochemical Features of Bacterial Isolates Obtained from Feed mill.......................40

Table 3: Antibiotic Resistance of Gram-negative bacterial isolates........................................41

Table 4: Antibiotic Resistance of Gram-positive bacterial isolates.........................................42

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 TABLE OF CONTENTS

TITLE PAGE ii
DECLARATION iii
CERTIFICATION iv
DEDICATION v
ACKNOWLEDGEMENT vi
LIST OF TABLES vii
TABLE OF CONTENTS viii
ABSTRACT xii
CHAPTER ONE 1
INTRODUCTION 1
1.1 Background of the Study 1

1.2 Statement of the Research Problem 3

1.3 Justification of the Study 5

1.4 Significance of the Study 7

1.5 Aim and Objectives of the Study 9

CHAPTER TWO 10
LITERATURE REVIEW 10
2.1 Overview of Antibiotics and Antimicrobial Resistance 10

2.1.1 Definition and Classes of Antibiotics 10

2.1.2 Mechanisms of Antibiotic Action 11

2.1.3 Global Emergence and Spread of Antibiotic Resistance 12

2.2 Mechanisms of Antibiotic Resistance in Bacteria 13

2.2.1 Genetic and Biochemical Mechanisms (Mutation, Efflux Pumps, Enzymatic

Degradation) 13

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 2.2.2 Horizontal Gene Transfer (Conjugation, Transformation, Transduction) 14

2.2.3 Role of Mobile Genetic Elements (Plasmids, Transposons) 15

2.3 Feed Mills as Reservoirs of Antibiotic-Resistant Bacteria 16

2.3.1 Microbial Contamination in Animal Feed 16

2.3.2 Sources and Entry Points of Resistant Bacteria in Feed Mills 17

2.3.3 Role of Cross-Contamination from Raw Materials and Equipment 18

2.4 Common Bacterial Isolates Found in Feed Mill Environments 20

2.4.1 Enterobacteriaceae (e.g., Escherichia coli, Salmonella spp.) 20

2.4.3 Opportunistic and Environmental Bacteria 21

2.5 Antibiotic Usage in Livestock and Its Impact on Feed-Associated Resistance 22

2.5.1 Use of Antibiotics as Growth Promoters and Prophylactics 22

2.5.2 Transfer of Resistance from Feed to Animal Gut Microbiota 22

2.5.3 Residual Antibiotics in Feed and Selective Pressure 23

2.6 Methods for Isolation and Identification of Bacteria from Feed 24

2.6.1 Sample Collection and Processing 24

2.6.2 Cultural, Biochemical, and Molecular Identification Techniques 25

2.6.3 Quality Assurance and Avoidance of False Negatives 25

2.7 Antimicrobial Susceptibility Testing (AST) 26

2.7.1 Principles of Disc Diffusion and Broth Dilution Methods 26

2.7.2 Interpretation of Results Based on CLSI/EUCAST Guidelines 27

2.7.3 Molecular Detection of Resistance Genes 28

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 2.8 Global and Regional Reports on Antibiotic Resistance in Animal Feed 28

2.8.1 Studies from Developed Countries (e.g., USA, EU, Canada) 28

2.8.2 Studies from Developing Countries (e.g., Nigeria, India, Brazil) 29

2.8.3 WHO and FAO Reports on Antimicrobial Resistance Surveillance 30

CHAPTER THREE 32
MATERIALS AND METHODS 32
3.1 Sterilization of Materials 32

3.2 Study Area 32

3.3 Preparation of Media 32

3.3.1 Preparation of Nutrient Agar (NA) 32

3.3.2 Preparation of Eosin Methylene Blue Agar Media 33

3.3.3 Preparation of Salmonella Shigella Agar (SSA) 33

3.4. Sampling Procedure 33

3.5 Physicochemical Analyses of Air Samples 34

3.6 Identification of the Isolated Bacteria 34

3.7.1. Morphological Identification Isolates 34

3.7.2 Gram Staining 35

3.7.3 Biochemical Tests 35

[Link] Catalase Test 35

[Link] Oxidase Test 36

[Link] Indole Test 36

[Link] Citrate Test 36

x
 [Link] Urease Test 37

3.6.1 Macroscopic Examination 37

3.6.2 Microscopic Examination 37

CHAPTER FOUR 38
RESULTS 38
4.1 Morphological Features of Isolates 38

4.2 Biochemical Features of Isolates 38

4.3 Antibiotic Resistance Pattern of Isolates 38

CHAPTER FIVE 43
DISCUSSION, CONCLUSION AND RECOMMENDATIONS 43
5.1 Discussion 43

5.2 Conclusion 46

5.3 Recommendations 47

REFERENCES 48

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 ABSTRACT

The increasing emergence of antimicrobial resistance (AMR) poses a global threat to public
health, particularly in the agricultural sector where the use of antibiotics in animal feed has
contributed to the spread of resistant microorganisms. This study was conducted to isolate
and screen bacteria from a feed mill environment in order to assess their antibiotic resistance
profiles and potential public health implications. The aim was to determine the types of
bacterial genera present in feed mill samples and evaluate their resistance to commonly used
antibiotics. Standard microbiological techniques were employed, including the collection of
feed mill samples, inoculation on selective and differential media, and identification of
bacterial isolates at the genus level using colonial morphology, Gram staining, and
biochemical characterization. Antibiotic susceptibility testing was carried out using the disc
diffusion method against a panel of antibiotics widely applied in veterinary and clinical
practice. The results revealed the presence of diverse bacterial genera, including Bacillus,
Pseudomonas, Klebsiella, Lactobacillus, Staphylococcus, and Enterobacter, indicating that
feed mills can serve as reservoirs of both environmental and opportunistic pathogens.
Antibiotic sensitivity patterns demonstrated variable resistance across isolates, with notable
resistance to ampicillin, tetracycline, and chloramphenicol, while gentamicin and
ciprofloxacin showed comparatively higher activity against most isolates. These findings
highlight the role of feed mills as potential sources of resistant bacteria that could be
transferred through the food chain to humans and animals, thus aggravating the spread of
AMR. In conclusion, the study underscores the need for routine microbial surveillance in
feed mills, judicious use of antibiotics in agriculture, and the adoption of alternative growth-
promoting strategies such as probiotics and plant-based additives. By addressing these
concerns, the risks of antibiotic resistance dissemination from agricultural sources to clinical
settings can be significantly reduced.

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CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

Antibiotics have revolutionized modern medicine and veterinary practice by serving as

effective tools for the prevention and treatment of infectious diseases caused by bacteria.

However, the excessive and indiscriminate use of antibiotics in both human healthcare and

animal husbandry has led to a growing global concern: the emergence and spread of

antibiotic-resistant bacteria (ARB). The phenomenon of antibiotic resistance occurs when

bacteria develop mechanisms to withstand the effects of antimicrobial agents that once could

eliminate them, rendering many first-line treatments ineffective and leading to prolonged

illness, higher healthcare costs, and increased mortality (Van Boeckel et al., 2020). While

clinical misuse is a significant driver of this problem, increasing attention is now being

directed towards non-clinical sources such as the food production chain, particularly animal

feeds, which serve as potential reservoirs and vectors for antibiotic-resistant microorganisms

(Tiseo et al., 2020).

In livestock production systems, feed mills are critical hubs for the preparation and

distribution of animal feeds. These facilities process various feed ingredients such as grains,

soybean meal, additives, and sometimes antibiotics or growth-promoting substances into a

composite meal that supports the nutritional needs of poultry, cattle, swine, and other farm

animals. In many regions, especially in low- and middle-income countries, antibiotics are

frequently incorporated into animal feed, either for prophylactic use, disease treatment, or to

enhance feed conversion efficiency and animal growth rates (FAO, 2021). Such practices,

often unregulated, contribute significantly to the selective pressure that promotes the
 2

development and proliferation of antibiotic-resistant bacteria in the agroecosystem. Feed

mills, therefore, represent an important environmental niche where bacterial contamination,

persistence, and horizontal gene transfer of resistance determinants may occur (Khine et al.,

2021).

Several studies have highlighted the presence of pathogenic and opportunistic bacteria, such

as Escherichia coli, Salmonella spp., Klebsiella spp., Enterococcus spp., and Pseudomonas

spp., in animal feed and feed ingredients. These bacteria not only pose a direct risk to animal

health but also have the potential to be transferred along the food chain to humans, especially

when meat, milk, or eggs from colonized animals are consumed. Of particular concern is the

detection of multidrug-resistant (MDR) strains in feed and feed-processing environments,

which could compromise therapeutic outcomes in veterinary and human medicine

(Ndahetuye et al., 2020). Feed mill workers are also at risk of occupational exposure to

aerosolized or contact-transmitted resistant microbes, thus acting as intermediaries in the

chain of resistance transmission (Sahoo et al., 2021).

The microbiological safety of animal feeds, particularly in countries like Nigeria where

regulatory enforcement is limited, is often overlooked in public health discourse. Many local

feed mills operate with minimal biosafety standards and limited quality control during raw

material procurement, storage, handling, and processing. This situation presents ideal

conditions for microbial contamination and the propagation of antibiotic resistance genes

(ARGs) via mobile genetic elements such as plasmids, integrons, and transposons (Thanner

et al., 2016). Moreover, contaminated feed can act as a long-term reservoir for resistance

traits that can spread through direct animal contact, manure application on agricultural fields,

or contamination of water systems, thus exacerbating environmental dissemination of

resistance.
 3

Understanding the bacterial profiles of feed mill environments and their resistance patterns is

therefore essential for developing surveillance strategies, guiding antibiotic stewardship

programs, and establishing feed safety protocols. In addition to traditional culture-based

microbiological methods, modern molecular techniques such as PCR and sequencing of

resistance genes are increasingly being employed to uncover the complex ecology of

resistance within feed systems. Despite these advancements, there remains a paucity of

localized research investigating the microbial contamination of animal feeds in Nigerian

contexts, particularly with regard to resistance profiling and its implications for public health

and food safety.

This study is designed to fill that knowledge gap by isolating and identifying bacterial strains

from a selected feed mill environment and evaluating their resistance to commonly used

antibiotics. By doing so, the research aims to contribute to the growing body of evidence on

non-clinical drivers of antimicrobial resistance (AMR) and highlight the urgent need for

integrated “One Health” approaches that encompass human, animal, and environmental

health sectors in tackling this global crisis (World Health Organization \[WHO], 2021).

1.2 Statement of the Research Problem

The rising prevalence of antibiotic resistance among bacterial pathogens poses one of the

most serious threats to public health in the 21st century. While antibiotic misuse in clinical

and veterinary medicine has long been recognized as a major driver of resistance, emerging

evidence indicates that the agricultural sector especially animal feed production and

distribution is also a significant contributor to the global antimicrobial resistance (AMR)

crisis (FAO, 2021; Tiseo et al., 2020). In Nigeria and other developing countries, local feed

mills often operate with minimal oversight, poor hygiene practices, and insufficient

regulatory enforcement regarding the use of antibiotics in feed formulations. This creates a
 4

conducive environment for microbial contamination and selective pressure that drives the

emergence and persistence of resistant bacteria in feed products and processing environments

(Khine et al., 2021).

Despite the potential public health risks, there is a notable scarcity of empirical data on the

microbiological safety of animal feeds in Nigeria, particularly regarding the presence of

multidrug-resistant (MDR) bacterial isolates. Most available studies have focused on clinical

samples or livestock waste, leaving a critical gap in understanding the microbiota associated

with feed mill operations. Furthermore, there is limited surveillance and inadequate

documentation of the resistance profiles of bacteria isolated from feed mills, especially in

rural or semi-urban settings where subsistence and commercial livestock farming heavily rely

on locally produced feeds. This lack of information undermines national and global efforts to

mitigate the spread of resistance genes across the food chain and into the broader

environment (Thanner et al., 2016).

Adding to the complexity is the widespread, and often undocumented, practice of including

antibiotics in animal feed as growth promoters or prophylactic agents. These practices not

only facilitate the proliferation of resistant bacteria but also encourage the horizontal transfer

of resistance genes via mobile genetic elements such as plasmids, integrons, and transposons

(Ndahetuye et al., 2020). Bacteria such as Escherichia coli, Salmonella spp., Klebsiella spp.,

and Pseudomonas spp. have been increasingly isolated from feed and are frequently found to

harbor resistance to multiple classes of antibiotics, including β-lactams, tetracyclines,

sulfonamides, and fluoroquinolones (Sahoo et al., 2021). These MDR organisms can be

transmitted to animals, humans, and the environment, thereby establishing a cycle of

resistance amplification with serious implications for food safety and public health.

The absence of comprehensive local studies evaluating the bacterial composition of feed mill

environments and their resistance phenotypes hinders the formulation of evidence-based

 5

policies and effective intervention strategies. In this context, it becomes imperative to isolate

and characterize the bacterial flora from feed mill environments and assess their antibiotic

susceptibility profiles. Understanding these parameters will provide critical insights into the

potential role of feed mills as reservoirs and transmission pathways for antibiotic-resistant

bacteria, and contribute to designing more robust biosecurity, monitoring, and stewardship

frameworks within the agricultural sector.

This study, therefore, addresses a pressing research gap by investigating the presence and

resistance profiles of bacterial isolates obtained from a feed mill. It seeks to generate baseline

data that can inform public health interventions, support antimicrobial stewardship, and align

with global “One Health” strategies aimed at controlling the spread of antimicrobial

resistance across human, animal, and environmental interfaces (WHO, 2021).

1.3 Justification of the Study

The alarming global rise in antimicrobial resistance (AMR) has intensified the need for

multidisciplinary research focused on identifying and mitigating potential sources of resistant

microorganisms. While considerable attention has been given to clinical and livestock

environments, less emphasis has been placed on feed mills despite their central role in the

animal production industry and their potential to serve as significant reservoirs and vectors

for the dissemination of resistant bacteria (FAO, 2021; Van et al., 2020). In countries such as

Nigeria, where regulatory oversight of feed production is often lax and routine

microbiological surveillance is lacking, feed mills may act as unnoticed hotspots for the

emergence and spread of multidrug-resistant (MDR) organisms within the food chain and

surrounding environment. The study is justified by this oversight and the urgent need to

address the knowledge gap that exists in this domain.

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Animal feeds are not only critical to livestock nutrition but also act as direct contact materials

that may introduce pathogens into animal systems, particularly when feeds are contaminated

by bacteria resistant to commonly used antibiotics. Such contamination can occur at various

points during processing, packaging, storage, or handling, especially in feed mills where

hygiene standards are suboptimal. The frequent and often unregulated inclusion of antibiotics

as growth promoters or prophylactics in feed production exerts selective pressure, thereby

encouraging the development and persistence of antibiotic-resistant bacteria (Ndahetuye et

al., 2020). These resistant microbes can subsequently be transmitted to animals, humans

(especially farm workers and consumers), and natural ecosystems, resulting in a complex and

dangerous public health issue that aligns with the “One Health” perspective of AMR

surveillance (WHO, 2021).

Furthermore, the identification and characterization of antibiotic-resistant bacteria from feed

mills are critical for developing context-specific interventions, including stricter regulatory

guidelines, improved feed processing hygiene, and rational use of antibiotics in livestock

production. In Nigeria, where livestock farming forms a major component of food security

and economic livelihoods, ensuring the microbiological quality and safety of feed is vital for

sustainable animal husbandry and public health protection. Currently, there is limited data

available on the bacterial contaminants of feed mills in Nigeria and even fewer studies that

explore their antimicrobial susceptibility patterns. This scarcity of localized information

undermines efforts to build evidence-based policy frameworks aimed at combatting AMR in

agricultural settings.

By isolating and identifying bacterial strains from feed mill environments and assessing their

resistance profiles, this study will contribute significantly to the limited body of knowledge

on AMR in non-clinical settings in Nigeria. The outcomes of the research can serve as a

baseline for future epidemiological surveillance, inform antimicrobial stewardship programs

 7

in agriculture, and support advocacy for the prudent use of antibiotics in feed formulations.

Moreover, the study aligns with the global agenda of minimizing the spread of antibiotic

resistance across human, animal, and environmental interfaces, thereby supporting broader

public health goals (O’Neill, 2020).

In summary, this study is both timely and essential. It addresses a critical blind spot in AMR

research, provides relevant data for local and global stakeholders, and supports the

development of targeted interventions that will contribute to the overall goal of controlling

the spread of antibiotic-resistant bacteria in the food and agricultural sectors.

1.4 Significance of the Study

The significance of this study lies in its potential to generate critical scientific knowledge that

addresses a growing global health concern: antimicrobial resistance (AMR). By focusing on

bacterial isolates obtained from feed mills an often-overlooked point in the food production

and animal husbandry chain this research provides vital insights into how antibiotic-resistant

bacteria may enter and circulate within the agricultural ecosystem. In developing nations like

Nigeria, where there is limited regulation and surveillance of antimicrobial use in the feed

industry, this study offers a timely and necessary contribution to public health, veterinary

medicine, and environmental microbiology.

One of the most pressing implications of antibiotic use in feed mills is the inadvertent

selection and propagation of resistant strains of bacteria such as Escherichia coli, Salmonella

spp., and Staphylococcus aureus. These bacteria can be transmitted to animals through feed,

and subsequently to humans via direct contact, contaminated meat, or environmental

pathways. By isolating and characterizing bacterial strains from feed mills and evaluating

their resistance profiles to commonly used antibiotics, this study helps in identifying possible

reservoirs of resistant pathogens within the food chain. The data generated will be
 8

indispensable for informing local antimicrobial stewardship programs and advocating for

policy changes that promote the prudent use of antibiotics in livestock production (FAO,

2021; WHO, 2022).

Furthermore, this study provides a scientific basis for routine microbial quality assessment of

animal feeds, a practice that is currently rare or inadequately enforced in many low- and

middle-income countries. The findings can help feed manufacturers improve hygiene

practices, identify contamination hotspots in the production process, and adopt non-antibiotic

alternatives such as probiotics or phytogenic additives. For veterinary professionals and

livestock farmers, this study enhances understanding of how contaminated feeds contribute to

infections in animals and therapeutic failures due to resistance. Ultimately, it contributes to

animal welfare, sustainable agriculture, and economic efficiency in livestock production.

The study also holds significance in the academic and research domain, as it contributes to

the limited body of literature on antibiotic resistance in non-clinical environments such as

feed mills in sub-Saharan Africa. It creates a foundation for future investigations and

multidisciplinary collaborations that intersect microbiology, veterinary science, agriculture,

and environmental health. For policymakers and public health authorities, the results will

serve as evidence for implementing regulatory frameworks aimed at controlling the use of

antibiotics in feed and mitigating the spread of resistance genes across ecological boundaries.

On a broader scale, the study supports the “One Health” approach by addressing antibiotic

resistance at the interface of human, animal, and environmental health. This aligns with

global AMR strategies proposed by institutions such as the World Health Organization

(WHO), the Food and Agriculture Organization (FAO), and the World Organisation for

Animal Health (WOAH), all of which emphasize the importance of coordinated action across

sectors. By offering concrete data and practical recommendations, this research contributes
 9

meaningfully to these global efforts and underscores the necessity of localized solutions to

what is ultimately a global crisis.

1.5 Aim and Objectives of the Study

Aim of the Study

The primary aim of this study is to isolate, identify, and determine the antibiotic resistance

profiles of bacterial strains obtained from feed mill environments in order to evaluate the

potential role of feed as a reservoir and transmission route for antimicrobial-resistant

pathogens.

Specific Objectives

1. To isolate and culture bacterial species from samples collected within a feed mill

environment, including feed, equipment surfaces, raw materials, and surrounding areas.

2. To identify the isolated bacterial strains using standard microbiological and biochemical

methods, and where applicable, molecular characterization techniques.

3. To determine the antibiotic susceptibility profiles of the identified bacterial isolates using

standardized antimicrobial susceptibility testing protocols such as the Kirby-Bauer disc

diffusion method.

4. To assess the prevalence and distribution of antibiotic-resistant bacteria in feed mill

samples, particularly those resistant to commonly used veterinary and human antibiotics (e.g.,

tetracycline, ampicillin, ciprofloxacin, gentamicin).

5. To evaluate the potential public health risks associated with the dissemination of resistant

bacteria through animal feed, and suggest appropriate control or mitigation strategies.
 10

6. To provide baseline data that may inform policy development and antimicrobial

stewardship in the animal feed industry, especially in regions with limited surveillance

infrastructure.
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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Overview of Antibiotics and Antimicrobial Resistance

2.1.1 Definition and Classes of Antibiotics

Antibiotics are a group of naturally occurring, semi-synthetic, or synthetic compounds that

are capable of killing or inhibiting the growth of microorganisms, particularly bacteria

(Khoshbakht et al., 2020). The term "antibiotic" was first used in the 1940s and originally

referred to substances produced by microorganisms that inhibited the growth of other

microbes. However, over time, it has expanded to include synthetic agents with similar

functions. Antibiotics are a cornerstone of modern medicine and have been instrumental in

treating bacterial infections, improving surgical outcomes, and enabling complex therapeutic

interventions such as chemotherapy and organ transplantation (World Health Organization

[WHO], 2021).

Antibiotics are broadly classified based on their chemical structure, mechanism of action, and

spectrum of activity. Chemically, antibiotics can be grouped into classes such as β-lactams

(e.g., penicillins, cephalosporins, carbapenems), aminoglycosides (e.g., gentamicin,

streptomycin), macrolides (e.g., erythromycin, azithromycin), tetracyclines, fluoroquinolones

(e.g., ciprofloxacin), sulfonamides, and glycopeptides (e.g., vancomycin) (Browning et al.,

2022). The classification according to mechanism of action includes inhibitors of cell wall

synthesis (β-lactams and glycopeptides), inhibitors of protein synthesis (aminoglycosides,

macrolides, tetracyclines), inhibitors of DNA synthesis (fluoroquinolones), and metabolic

antagonists (sulfonamides and trimethoprim) (Ferdous et al., 2023).

 12

Furthermore, antibiotics are categorized based on their spectrum of activity as broad-

spectrum (active against both Gram-positive and Gram-negative bacteria) or narrow-

spectrum (targeting specific bacterial groups). This categorization is essential in clinical and

veterinary medicine, including the feed and livestock industries, where broad-spectrum

antibiotics are frequently administered for disease prevention and growth promotion (Osei

Sekyere and Mensah, 2021). However, indiscriminate and non-therapeutic usage of these

antibiotics has fueled the global crisis of antimicrobial resistance.

2.1.2 Mechanisms of Antibiotic Action

The effectiveness of antibiotics hinges on their ability to target specific components of

bacterial cells that are either absent or significantly different in human cells, minimizing harm

to the host. Each class of antibiotics exerts its antibacterial effects through distinct

mechanisms. For instance, β-lactam antibiotics such as penicillin inhibit the synthesis of

peptidoglycan, an essential component of the bacterial cell wall, leading to cell lysis and

death (Li et al., 2021). Glycopeptides such as vancomycin similarly target cell wall synthesis

but through a different binding mechanism. Aminoglycosides, macrolides, and tetracyclines

inhibit protein synthesis by binding to bacterial ribosomal subunits, thereby preventing

translation and inhibiting bacterial growth (Olaitan et al., 2022).

Fluoroquinolones act by inhibiting DNA gyrase and topoisomerase IV, enzymes essential for

bacterial DNA replication and repair, leading to DNA damage and cell death. Sulfonamides

and trimethoprim disrupt folic acid synthesis, an essential pathway in bacterial metabolism,

thereby exerting bacteriostatic effects (Gonzalez et al., 2020). Some antibiotics, like

polymyxins, target the bacterial cell membrane, causing loss of membrane integrity and

leakage of cellular contents.

 13

These mechanisms not only define the clinical utility of antibiotics but also influence the

selection pressures that drive the emergence of resistance. In feed mill environments, where

multiple classes of antibiotics may be used, either directly in feed or indirectly through

contamination, bacteria are exposed to a complex array of antimicrobial agents. This

environment serves as a selective ground for the emergence of resistant strains and facilitates

the exchange of resistance genes (Ahmed et al., 2022).

2.1.3 Global Emergence and Spread of Antibiotic Resistance

Antimicrobial resistance (AMR) has emerged as one of the most pressing global public health

challenges of the 21st century. It refers to the ability of microorganisms, particularly bacteria,

to withstand the effects of antibiotics that once effectively inhibited or killed them. The

widespread and often inappropriate use of antibiotics in human medicine, veterinary practice,

and agriculture has accelerated the development of resistant strains across various ecosystems

(Mantegazza et al., 2021). The use of antibiotics in animal feed, especially as growth

promoters and prophylactics, has been identified as a major contributor to the global AMR

crisis (WHO, 2021).

Bacteria can acquire resistance through spontaneous genetic mutations or more commonly

through horizontal gene transfer (HGT), involving mechanisms such as conjugation,

transformation, and transduction. Resistance genes are frequently carried on mobile genetic

elements like plasmids, transposons, and integrons, which can spread between bacteria of the

same or different species (Cheng et al., 2023). Once introduced into a microbial community,

resistant strains can persist and proliferate, especially under selective pressure from continued

antibiotic exposure.

Globally, antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus

(MRSA), extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, carbapenem-

 14

resistant Enterobacteriaceae (CRE), and vancomycin-resistant Enterococci (VRE) have been

implicated in numerous outbreaks and are associated with high morbidity, mortality, and

healthcare costs (O’Neill, 2022). In agricultural systems, resistant bacteria from feed mills

can enter the food chain, contaminate the environment, and even reach human populations

through direct contact with animals, consumption of animal products, or exposure to

contaminated water (Ezenduka et al., 2021).

Studies from developing countries, including Nigeria, have shown alarmingly high levels of

antibiotic-resistant bacteria in animal feeds, feed mill environments, and poultry farms, with

resistance profiles often including multi-drug resistance (MDR) (Adebowale et al., 2020;

Odetokun et al., 2021). This global and regional evidence underscores the urgent need for

coordinated surveillance, prudent antibiotic use policies, and robust regulatory frameworks to

curb the spread of AMR from feed sources.

2.2 Mechanisms of Antibiotic Resistance in Bacteria

2.2.1 Genetic and Biochemical Mechanisms (Mutation, Efflux Pumps, Enzymatic

Degradation)

Antibiotic resistance in bacteria arises through a variety of genetic and biochemical

mechanisms that render antimicrobial agents ineffective. One of the primary mechanisms is

spontaneous mutation, which can alter the target site of an antibiotic, diminishing its ability to

bind and exert its action. For instance, mutations in genes encoding ribosomal proteins or

RNA polymerase can reduce susceptibility to macrolides or rifampicin, respectively (Murray

et al., 2022). These mutations are often selected under antibiotic pressure and can quickly

become fixed in bacterial populations.

Efflux pumps constitute another vital biochemical mechanism, allowing bacteria to actively

transport antibiotics out of the cell. These transport proteins can be specific to a particular
 15

drug or have broad substrate specificity, thus contributing to multidrug resistance (MDR)

(Wang et al., 2021). Prominent among them are the Resistance-Nodulation-Division (RND)

family of efflux pumps in Gram-negative bacteria, such as AcrAB-TolC in Escherichia coli,

which extrudes tetracyclines, fluoroquinolones, and β-lactams (Li et al., 2020).

A third, extensively reported mechanism involves enzymatic degradation or modification of

antibiotics. β-lactamases, for example, hydrolyze the β-lactam ring of penicillins and

cephalosporins, rendering them inactive. Extended-spectrum β-lactamases (ESBLs) and

carbapenemases such as KPC (Klebsiella pneumoniae carbapenemase) and NDM (New Delhi

metallo-β-lactamase) have emerged as major public health threats due to their broad activity

against β-lactam antibiotics (Nwobodo et al., 2023; Osei Sekyere, 2020). Additionally,

aminoglycoside-modifying enzymes (AMEs) can acetylate, phosphorylate, or adenylate

aminoglycosides, reducing their binding to ribosomal targets (Ahmed et al., 2022).

These genetic and biochemical resistance mechanisms not only compromise therapeutic

outcomes but also complicate clinical management by necessitating the use of last-resort

antibiotics, which may have higher toxicity or limited availability.

2.2.2 Horizontal Gene Transfer (Conjugation, Transformation, Transduction)

The rapid dissemination of antibiotic resistance among bacterial populations is largely driven

by horizontal gene transfer (HGT), a process through which genetic material is exchanged

between organisms without reproduction. Three major modes of HGT conjugation,

transformation, and transduction play pivotal roles in the spread of resistance genes in diverse

environments, including feed mills.

Conjugation involves the direct transfer of plasmids from one bacterial cell to another via

physical contact, often mediated by sex pili. Plasmids carrying resistance genes such as bla\

_TEM, bla\_CTX-M, and mcr-1 have been widely reported in Enterobacteriaceae isolated
 16

from animal feed and fecal matter (Alonso et al., 2022). This mechanism facilitates the rapid

spread of multidrug resistance, especially in densely populated microbial environments.

Transformation, in contrast, entails the uptake and incorporation of free DNA fragments from

the environment into a competent bacterium’s genome. Although less common in nature,

transformation can still significantly contribute to resistance gene dissemination, particularly

in naturally competent species like Streptococcus pneumoniae and Acinetobacter baumannii

(Zhu et al., 2021). Studies have shown that environmental DNA from feed and waste

materials can serve as reservoirs of resistance determinants (Abia et al., 2021).

Transduction, mediated by bacteriophages, allows for the accidental transfer of resistance

genes during viral replication cycles. Although often underestimated, phage-mediated gene

transfer has been implicated in the mobilization of resistance elements such as mecA and erm

genes (Matos et al., 2021). Given the widespread presence of phages in the environment,

especially in soil and animal waste, their contribution to resistance spread in feed mill

settings cannot be overlooked.

Overall, HGT facilitates the accumulation and co-selection of multiple resistance genes

within a single bacterial genome or plasmid, leading to complex resistance profiles that are

increasingly difficult to treat.

2.2.3 Role of Mobile Genetic Elements (Plasmids, Transposons)

Mobile genetic elements (MGEs), including plasmids, transposons, integrons, and insertion

sequences, are central to the mobility and persistence of antibiotic resistance genes in

bacterial populations. These elements serve as vectors that facilitate both intra- and inter-

species gene transfer, thus exacerbating the resistance crisis.

Plasmids, which are extrachromosomal DNA molecules capable of autonomous replication,

are perhaps the most notorious MGEs associated with resistance. They often carry multiple
 17

resistance genes in clusters, allowing for the simultaneous spread of resistance to several

antibiotics. For instance, plasmid-mediated colistin resistance (mcr-1) and carbapenem

resistance genes (bla\_NDM, bla\_KPC) have been isolated from E. coli and Klebsiella spp.

in poultry and feed mills, highlighting the agricultural sector’s contribution to resistance

dissemination (Singh et al., 2021).

Transposons, or "jumping genes," facilitate the movement of DNA segments between

plasmids and chromosomes. Composite transposons such as Tn3 and Tn7 can harbor multiple

resistance genes and can insert into a variety of genomic contexts, enhancing the adaptability

of bacterial hosts (Xie et al., 2020). Additionally, transposons often carry integron cassettes,

which serve as platforms for the integration and expression of additional gene segments.

Integrons, while not mobile by themselves, capture gene cassettes including antibiotic

resistance genes through site-specific recombination. Class 1 integrons are particularly

prevalent in clinical and environmental isolates and have been implicated in the spread of

multidrug resistance in Gram-negative bacteria from agricultural settings (Kariuki et al.,

2022).

The interconnectedness of these MGEs forms a complex genomic network that facilitates

rapid adaptation and survival in antimicrobial-laden environments like feed mills. Their

presence contributes significantly to the global health burden posed by antimicrobial

resistance, necessitating urgent surveillance and mitigation strategies.

2.3 Feed Mills as Reservoirs of Antibiotic-Resistant Bacteria

2.3.1 Microbial Contamination in Animal Feed

Animal feed is a critical component in livestock production, but it often serves as a vehicle

for microbial contamination, including antibiotic-resistant bacteria (ARB). Animal feed

ingredients, whether of plant or animal origin, are susceptible to contamination during

 18

harvesting, processing, storage, and transportation. This contamination is not only limited to

spoilage microorganisms but also includes pathogenic and opportunistic bacteria such as

Salmonella, Escherichia coli, Enterococcus, and Clostridium spp. (Bohm et al., 2020; Lee et

al., 2022). These organisms may carry genes that confer resistance to commonly used

antibiotics, and their presence in feed implies a potential risk for transmission into the animal

gastrointestinal system and, consequently, into the food chain.

Studies have shown that a variety of feedstuffs harbor multidrug-resistant bacteria (MDR),

particularly those intended for poultry and swine production (Zhang et al., 2021). Animal

feed products containing meat and bone meal, fishmeal, or blood meal tend to have higher

microbial loads compared to plant-based feed due to their high protein and moisture content,

which favor microbial growth (Park et al., 2021). Additionally, feed additives such as

antibiotics used as growth promoters have historically contributed to the selection and

proliferation of resistant bacterial strains within feed mill environments (Verschuuren et al.,

2022). Even after the ban on growth-promoting antibiotics in many countries, remnants of

these practices continue to impact microbial profiles in feed systems.

The use of poorly regulated or substandard feed further compounds the problem. In

developing countries, in particular, the microbial safety of animal feed is rarely monitored or

enforced, resulting in persistent microbial contamination and the possibility of antibiotic

resistance transfer through feed (Nworie et al., 2023). Hence, feed contamination not only

poses a threat to animal health and productivity but also presents a significant public health

hazard due to its role in maintaining reservoirs of ARB in livestock production chains.

2.3.2 Sources and Entry Points of Resistant Bacteria in Feed Mills

Feed mills act as nodes in the agro-industrial supply chain where raw materials from diverse

sources are collected, mixed, processed, and distributed. This complex operation provides
 19

multiple opportunities for the introduction and propagation of ARB. One of the primary entry

points of resistant bacteria is the raw materials themselves grains, oilseeds, and animal by-

products which may be contaminated during cultivation, harvesting, or processing (Singh et

al., 2022). Environmental exposure to manure, wastewater, or untreated biosolids often used

as fertilizers can introduce resistant strains into crops and, by extension, into feed mills.

In addition to raw materials, water used in various stages of feed preparation is another

important vector. Inadequate treatment or storage of water can introduce resistant bacteria,

particularly from sources polluted by agricultural runoff or effluents from pharmaceutical and

healthcare facilities (Aliyu et al., 2021). Workers, machinery, and vehicles entering feed mill

premises without proper biosecurity protocols also serve as mechanical carriers of resistant

microorganisms (Almeida et al., 2023).

Furthermore, studies have documented how poor hygiene practices and inadequate cleaning

schedules can cause the build-up of biofilms in feed mill equipment such as mixers,

conveyors, and silos (Park et al., 2021). These biofilms can harbor ARB and protect them

from disinfectants, making them persistent sources of contamination (Wu et al., 2022). Dust

particles generated during feed processing have also been found to carry resistant bacteria,

which can settle on equipment and raw materials, creating a cycle of recontamination

(Nguyen et al., 2020).

The cumulative effect of these entry points results in a feed mill microbiome that may contain

highly diverse and resistant bacterial populations. Such populations, if unchecked, can

continuously reintroduce ARB into animal feeds and ultimately to animals, posing a serious

challenge to antibiotic stewardship efforts in agriculture and food production.

 20

2.3.3 Role of Cross-Contamination from Raw Materials and Equipment

Cross-contamination in feed mills is a significant driver of the spread of ARB within the

facility and downstream into animal husbandry. The processing environment in a feed mill is

often complex and interconnected, with shared machinery and transportation systems used for

both contaminated and uncontaminated materials. This interconnectedness allows bacteria to

move from raw ingredients to finished feed through surface contact and air transmission (Xia

et al., 2021). For example, if a batch of feed contaminated with Enterococcus faecalis

containing vancomycin resistance genes is processed, and the equipment is not properly

sanitized before the next batch, resistant bacteria can be transferred to otherwise clean feed.

Cross-contamination is especially problematic when feed mills handle multiple types of feed

products for different animal species. Equipment used in grinding, pelleting, or mixing can

act as fomites that retain bacterial residues even after superficial cleaning (Moyaert et al.,

2023). Pelleting at sub-optimal temperatures may not be sufficient to eliminate

thermotolerant ARB, especially those embedded in protective matrices or biofilms on

equipment surfaces (Chen et al., 2021).

Additionally, inadequate segregation between clean and dirty zones within the mill

contributes to the unregulated movement of personnel and materials, which accelerates

microbial dispersion. Workers may carry resistant bacteria on their clothing, footwear, or

hands from one part of the facility to another, compounding the problem of environmental

contamination and increasing the probability of feed cross-contamination (Kabir et al., 2022).

The consequences of such cross-contamination are far-reaching. Animals that consume

contaminated feed may become asymptomatic carriers of ARB, which can then be shed into

the environment through feces. This can lead to contamination of soil, water, and crops,

effectively creating a feedback loop that perpetuates the antibiotic resistance cycle.

Moreover, humans handling animals or consuming their products (meat, milk, eggs) may be
 21

exposed to these ARB, thereby raising concerns about zoonotic transmission (Oloso et al.,

2021).

2.4 Common Bacterial Isolates Found in Feed Mill Environments

2.4.1 Enterobacteriaceae (e.g., Escherichia coli, Salmonella spp.)

Members of the Enterobacteriaceae family are frequently isolated from animal feed and feed

mill environments due to their ubiquity in fecal-contaminated raw materials and poor

hygienic practices. Escherichia coli (E. coli) is a prominent coliform bacterium commonly

used as an indicator of fecal contamination. Pathogenic strains, such as enterohemorrhagic

and enterotoxigenic E. coli, have been associated with animal infections and pose significant

public health risks when transmitted through the food chain (Singh et al., 2022). Salmonella

spp., especially Salmonella enterica, are widely recognized as feed-borne pathogens, capable

of surviving in dry feed ingredients and colonizing both animals and processing

environments. These organisms are particularly concerning due to their ability to develop

resistance to a wide range of antibiotics, including fluoroquinolones and extended-spectrum

beta-lactams (Akbar and Anal, 2020). The high microbial load in feed mills, particularly from

poorly processed or stored raw materials like poultry droppings, bone meal, and blood meal,

fosters the proliferation and persistence of these Gram-negative pathogens (Zhang et al.,

2021). Regular monitoring and microbial risk assessment are necessary to reduce the burden

of Enterobacteriaceae in feed production systems.

2.4.2 Gram-Positive Pathogens (e.g., Staphylococcus aureus, Enterococcus spp.)

Gram-positive bacteria, notably Staphylococcus aureus and Enterococcus species, are also

prevalent in feed mill environments and contribute significantly to the antibiotic resistance

pool. Staphylococcus aureus can persist on surfaces and in dust particles, making feed

handling equipment and storage bins common contamination points. Methicillin-resistant S.

 22

aureus (MRSA), in particular, has been isolated from livestock feed and associated facilities,

indicating a possible link between feed contamination and zoonotic transmission (Weese et

al., 2022). Similarly, Enterococcus faecalis and Enterococcus faecium, which naturally reside

in the intestinal tracts of animals, can contaminate feed during handling and packaging. These

bacteria are increasingly exhibiting resistance to vancomycin and linezolid, two critical

antibiotics used in human medicine (Hossain et al., 2023). The adaptive capabilities of Gram-

positive bacteria, coupled with selective pressures from antibiotic residues in feed, have

facilitated the emergence of multidrug-resistant (MDR) strains that are challenging to

eradicate in feed mills. Furthermore, their presence can be exacerbated by the use of

antibiotics as growth promoters in livestock, leading to continuous environmental exposure

(Aklilu and Ramanathan, 2021).

2.4.3 Opportunistic and Environmental Bacteria

In addition to well-known pathogens, feed mill environments also harbor a diverse range of

opportunistic and environmental bacteria, such as Pseudomonas, Acinetobacter, Bacillus, and

Klebsiella spp., which may not be primary pathogens but serve as reservoirs of resistance

genes. Pseudomonas aeruginosa, known for its intrinsic resistance and adaptive resistance

mechanisms, is frequently found in moist feed mill zones, including drainage pipes and

poorly ventilated areas. It possesses multiple efflux pumps and biofilm-forming abilities that

enhance its resistance to disinfectants and antimicrobials (Murray et al., 2022). Acinetobacter

baumannii, another notorious environmental bacterium, has been isolated from feed-related

surfaces and has shown high resistance to carbapenems and aminoglycosides, posing

challenges in nosocomial and veterinary settings alike (Ramirez et al., 2021). While Bacillus

spp. are often considered beneficial as probiotics in feed additives, certain strains such as

Bacillus cereus can produce enterotoxins and harbor mobile resistance genes (Chandel et al.,
 23

2023). These environmental isolates contribute to the resistome of the feed mill ecosystem

and facilitate horizontal gene transfer among coexisting bacterial populations. Their role in

the transmission of resistance elements emphasizes the need for comprehensive microbial

surveillance and the implementation of strict biosecurity protocols across feed production

chains.

2.5 Antibiotic Usage in Livestock and Its Impact on Feed-Associated Resistance

2.5.1 Use of Antibiotics as Growth Promoters and Prophylactics

The incorporation of antibiotics in livestock feed for non-therapeutic purposes, particularly as

growth promoters and prophylactic agents, has been a major contributor to the emergence and

dissemination of antibiotic-resistant bacteria in feed mill environments. Growth-promoting

antibiotics are administered at sub-therapeutic doses to enhance feed efficiency, stimulate

animal growth, and prevent disease outbreaks in intensive farming systems (Diarra and

Malouin, 2021). This widespread and routine application leads to the continuous exposure of

gut microbiota and environmental bacteria to antibiotics, creating ideal conditions for the

selection and proliferation of resistant strains. Prophylactic use, where antibiotics are

administered to entire herds or flocks regardless of individual health status, further

accelerates resistance development by applying uniform selective pressure across diverse

bacterial populations (Nhung et al., 2022). In regions with limited regulatory oversight, such

as parts of sub-Saharan Africa and Southeast Asia, antibiotics like tetracyclines, macrolides,

and beta-lactams are often overused or misused in animal feeds without veterinary guidance

(Van Cuong et al., 2021). These practices promote the survival of resistant bacteria, many of

which colonize feed processing environments and enter the food chain through contaminated

animal products.
 24

2.5.2 Transfer of Resistance from Feed to Animal Gut Microbiota

Feed-associated antibiotic-resistant bacteria can colonize the gastrointestinal tract of animals,

serving as vectors for the horizontal transmission of resistance genes. Upon ingestion,

resistant bacteria present in feed or on feed-contact surfaces may interact with commensal gut

microbes, facilitating the exchange of resistance determinants through plasmids, transposons,

and integrons (Zheng et al., 2020). This transfer can result in the emergence of multidrug-

resistant (MDR) gut flora, which may subsequently be shed into the environment via feces,

contributing to environmental contamination and persistence of resistance reservoirs. For

example, resistant E. coli and Salmonella strains from feed have been shown to colonize

poultry and swine intestinal tracts, leading to asymptomatic carriage or clinical infections in

animals and increased risk of zoonotic transmission to humans (Mughini-Gras et al., 2021).

The gut microbiome’s role as a reservoir and amplifier of resistance genes underscores the

complexity of the feed–animal–environment–human interface. Continuous exposure of the

gut microbiota to low levels of antibiotics from feed facilitates the selection of resistant

bacteria, which can subsequently be transferred to humans through the consumption of meat,

milk, or eggs, or through direct animal contact (Kempf et al., 2022).

2.5.3 Residual Antibiotics in Feed and Selective Pressure

Beyond the direct inclusion of antibiotics in feed, residual antimicrobial compounds either

due to carryover during feed production or through contaminated raw materials play a

significant role in maintaining selective pressure within feed mill ecosystems. Such residues,

even at trace concentrations, are sufficient to inhibit susceptible bacterial populations while

allowing resistant variants to thrive and dominate microbial communities (Fang et al., 2020).

Antibiotic residues can persist in feed components like bone meal, fish meal, and feather

meal, which are often derived from previously medicated animals. Furthermore, improper
 25

feed storage and poor decontamination practices of equipment and silos can result in the

accumulation and recirculation of antibiotic residues (Zhao et al., 2023). These residual

antibiotics exert continuous selection pressure, encouraging the proliferation of bacteria with

pre-existing resistance mechanisms such as efflux pumps, antibiotic-degrading enzymes, and

altered target sites. Studies have also shown that sub-inhibitory antibiotic concentrations can

enhance horizontal gene transfer rates and biofilm formation, thereby amplifying the spread

of resistance determinants within feed mill microbial communities (Yin et al., 2021). Thus,

the presence of antibiotic residues in feed, even when unintentional, represents a persistent

and underestimated risk factor in the broader context of antimicrobial resistance.

2.6 Methods for Isolation and Identification of Bacteria from Feed

2.6.1 Sample Collection and Processing

Accurate isolation of bacteria from animal feed begins with systematic and aseptic sample

collection and handling to ensure the representativeness and integrity of the microbial

community present. Feed samples should be collected from various points within the feed

mill, including raw materials, mixers, hoppers, conveyors, storage silos, and final feed

products to capture the full microbial profile (Barros et al., 2021). Standard operating

procedures recommend the use of sterile containers and tools, and samples should be kept

under refrigeration (4–8 °C) and processed within 24 hours to prevent changes in bacterial

populations due to storage conditions (Medeiros et al., 2023). Homogenization of feed

samples is achieved by mixing in buffered peptone water or phosphate-buffered saline

followed by serial dilution. These suspensions are used to inoculate selective and non-

selective media to culture diverse bacterial groups. Feed with high fat or particulate content

may require additional steps such as filtration or centrifugation to remove inhibitors and

enhance bacterial recovery (Vázquez et al., 2022). In addition, enrichment broths are often
 26

employed prior to plating, particularly for recovering fastidious or low-abundance bacteria

like Salmonella spp., which may be present in low numbers or suppressed by more dominant

microbes (Islam et al., 2021).

2.6.2 Cultural, Biochemical, and Molecular Identification Techniques

A combination of culture-based and molecular methods is typically used to identify bacterial

isolates from feed, enabling both broad screening and precise species-level identification.

After sample inoculation onto media such as MacConkey agar, nutrient agar, and blood agar,

colony morphology, hemolysis patterns, and pigmentation are observed as preliminary

identification steps (Yasir et al., 2022). Gram staining and microscopic examination are used

to differentiate Gram-positive from Gram-negative bacteria. Biochemical tests such as

catalase, oxidase, urease, indole production, triple sugar iron (TSI), citrate utilization, and

API 20E kits provide further phenotypic identification (Doyle and Erickson, 2020). However,

due to limitations in sensitivity and specificity, molecular methods like polymerase chain

reaction (PCR), 16S rRNA gene sequencing, and multiplex PCR are increasingly applied to

detect and differentiate bacteria, especially those with similar phenotypic traits or those

present in mixed cultures (Rafiee et al., 2022). Real-time PCR offers rapid, quantitative

detection of specific resistance genes such as blaTEM, tetA, and mecA, while whole-genome

sequencing (WGS) provides detailed information on virulence factors and resistance gene

clusters (Zhou et al., 2021). The integration of both classical and molecular techniques

ensures robust and accurate bacterial identification in complex feed samples.

2.6.3 Quality Assurance and Avoidance of False Negatives

The accuracy and reliability of bacterial isolation and identification procedures in feed

microbiology hinge on stringent quality assurance measures and methodological vigilance to

prevent false negatives or misleading results. False negatives may arise from improper
 27

sampling, insufficient sample volume, poor culture conditions, or interference from

antimicrobial residues or feed additives that inhibit microbial growth (Aslam et al., 2021). To

mitigate these issues, internal controls, parallel negative and positive controls, and duplicate

sampling are used throughout the processing and analysis stages. Quality assurance protocols

must include routine equipment sterilization, media performance verification, and calibration

of instruments such as thermocyclers, pH meters, and incubators (Gong et al., 2020). Regular

training of laboratory personnel and adherence to standardized microbiological protocols

such as those recommended by ISO 6579 for Salmonella and ISO 4833 for total aerobic

count are also essential. Furthermore, employing pre-enrichment and selective enrichment

steps improves the recovery of slow-growing or sublethally injured bacteria (Mughini-Gras

et al., 2022). Incorporating molecular validation techniques such as internal amplification

controls (IACs) during PCR minimizes the risk of false negatives due to inhibitors in feed

matrices. Ultimately, a comprehensive quality assurance framework is critical for ensuring

the fidelity of feed microbiological analysis and the accurate detection of antibiotic-resistant

bacteria.

2.7 Antimicrobial Susceptibility Testing (AST)

2.7.1 Principles of Disc Diffusion and Broth Dilution Methods

Antimicrobial Susceptibility Testing (AST) plays a pivotal role in determining the efficacy of

antibiotics against bacterial isolates, helping to guide treatment choices and monitor

resistance patterns. The disc diffusion method (Kirby-Bauer test) is a widely used phenotypic

assay based on the principle of diffusion of antibiotics from impregnated paper discs into an

agar medium, creating a concentration gradient. Bacterial isolates are uniformly inoculated

on Mueller-Hinton agar, and discs containing specific concentrations of antibiotics are placed

on the surface. After incubation at 35°C for 16–24 hours, the diameter of the inhibition zone
 28

around each disc is measured and compared to standardized reference values to classify

bacteria as susceptible, intermediate, or resistant (Magiorakos et al., 2022). In contrast, broth

dilution methods comprising macro and microdilution techniques determine the Minimum

Inhibitory Concentration (MIC), the lowest concentration of an antibiotic that visibly inhibits

bacterial growth. Serial dilutions of antibiotics are prepared in broth and inoculated with a

standardized bacterial suspension. The MIC is interpreted as the lowest drug concentration

where no visible turbidity is observed after incubation (Jorgensen and Ferraro, 2020). While

disc diffusion is simple and cost-effective for routine use, broth dilution provides quantitative

data useful for pharmacokinetics/pharmacodynamics (PK/PD) modeling and advanced

surveillance studies.

2.7.2 Interpretation of Results Based on CLSI/EUCAST Guidelines

The interpretation of AST results must adhere to internationally accepted standards to ensure

consistency, comparability, and clinical relevance. Two leading authorities Clinical and

Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial

Susceptibility Testing (EUCAST) provide interpretive breakpoints and methodological

guidelines. These standards define susceptibility categories (susceptible, intermediate,

resistant) based on zone diameter or MIC thresholds that correlate with clinical outcomes and

achievable drug concentrations at infection sites (CLSI, 2023; EUCAST, 2022). CLSI

primarily uses fixed MIC and inhibition zone breakpoints, while EUCAST applies dose-

specific breakpoints, integrating PK/PD relationships more extensively. Both organizations

regularly update their guidelines to account for emerging resistance mechanisms and changes

in drug efficacy. AST results are interpreted using established breakpoint tables, and quality

control strains like Escherichia coli ATCC 25922 or Staphylococcus aureus ATCC 25923 are

included in each batch to validate test performance. Laboratories must ensure that testing
 29

conditions, media composition, inoculum density, and incubation times conform strictly to

these standards to avoid errors in classification. Accurate interpretation of AST results is

critical for infection control, resistance surveillance, and rational antimicrobial use in both

clinical and veterinary microbiology.

2.7.3 Molecular Detection of Resistance Genes

Phenotypic susceptibility tests can be complemented by molecular techniques that directly

identify specific resistance genes, offering rapid and precise detection, especially in the

context of multidrug resistance (MDR) or when phenotypic methods yield ambiguous results.

Polymerase Chain Reaction (PCR), multiplex PCR, and real-time PCR are commonly used to

amplify and detect genes encoding resistance determinants such as blaTEM, blaCTX-M,

tetA, mecA, vanA, and aac(6')-Ib (Rajendran et al., 2022). These methods are highly

sensitive and specific, capable of detecting low-copy gene targets even in mixed microbial

communities. In addition, molecular assays can reveal resistance mechanisms such as

extended-spectrum β-lactamase (ESBL) production, methicillin resistance, or plasmid-

mediated colistin resistance (e.g., mcr genes) that may not be apparent phenotypically (Zhou

et al., 2021). Whole genome sequencing (WGS) and DNA microarrays provide a broader

view by simultaneously identifying multiple resistance genes, mobile genetic elements, and

associated virulence factors. Despite their accuracy, molecular tests are not a substitute for

AST since gene presence does not always equate to gene expression or phenotypic resistance.

Hence, integrating molecular data with phenotypic results offers a comprehensive

understanding of bacterial resistance profiles, particularly important in One Health

surveillance of animal-origin bacteria in feed environments.

 30

2.8 Global and Regional Reports on Antibiotic Resistance in Animal Feed

2.8.1 Studies from Developed Countries (e.g., USA, EU, Canada)

In developed regions such as the United States, European Union, and Canada, antibiotic

resistance in animal feed has received significant attention due to its implications for food

safety, environmental health, and public health. In the United States, surveillance programs

like the National Antimicrobial Resistance Monitoring System (NARMS) have tracked

antimicrobial resistance (AMR) trends in bacteria from animals, retail meats, and humans

since 1996. NARMS data indicate the presence of multidrug-resistant (MDR) strains of

Salmonella, Campylobacter, and Escherichia coli in livestock and feed samples, with

resistance to critically important antibiotics such as fluoroquinolones and cephalosporins

(Tadesse et al., 2022). Similarly, in the European Union, the European Food Safety Authority

(EFSA) and European Medicines Agency (EMA) jointly reported significant reductions in

the use of antimicrobials in livestock since the implementation of restrictive policies,

including the 2006 ban on antibiotics as growth promoters. Despite these improvements,

surveillance reveals persistent resistance to tetracyclines, sulfonamides, and aminoglycosides

among enteric bacteria isolated from feed and animals (EMA and EFSA, 2022). Canada’s

Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) has

identified resistance trends in both imported and domestically produced feeds, particularly in

E. coli isolates, often linked to historical overuse of antimicrobials in poultry and swine

farming (Government of Canada, 2023). These surveillance systems have played a critical

role in informing policies and interventions aimed at mitigating AMR spread in food

production systems.
 31

2.8.2 Studies from Developing Countries (e.g., Nigeria, India, Brazil)

In developing nations, surveillance and reporting of antibiotic resistance in animal feed are

less standardized but increasingly recognized due to rising antimicrobial usage in livestock

and inadequate regulatory enforcement. In Nigeria, studies have demonstrated widespread

contamination of animal feed with antibiotic-resistant bacteria, particularly

Enterobacteriaceae, Staphylococcus spp., and Pseudomonas spp., often resistant to

tetracycline, ampicillin, and erythromycin (Olowe et al., 2021; Ayandiran et al., 2022).

Factors contributing to this include uncontrolled access to veterinary drugs, use of antibiotics

as prophylactics and growth enhancers, and poor feed manufacturing practices. In India, a

leading producer and consumer of livestock, high levels of antimicrobial residues and MDR

pathogens in poultry and cattle feed have been reported. A study by Van Boeckel et al.

(2021) showed the prevalence of ESBL-producing E. coli and resistance genes such as

blaCTX-M and tetA in animal feeds, linked to high veterinary antibiotic consumption.

Similarly, in Brazil a major exporter of meat products investigations have identified

Salmonella enterica and E. coli strains in animal feeds exhibiting resistance to third-

generation cephalosporins and fluoroquinolones (Oliveira et al., 2023). However, monitoring

systems in these regions are often fragmented, with limited governmental oversight. The lack

of harmonized AMR surveillance frameworks has hindered effective policy responses,

although recent initiatives funded by international organizations are beginning to address

these gaps.

2.8.3 WHO and FAO Reports on Antimicrobial Resistance Surveillance

The World Health Organization (WHO), in collaboration with the Food and Agriculture

Organization (FAO) and the World Organisation for Animal Health (WOAH), has

emphasized the global threat of antimicrobial resistance across human, animal, and
 32

environmental interfaces. The Global Action Plan on AMR launched by WHO in 2015

identified food-producing animals and their feed as critical points of intervention. Through

the Global Antimicrobial Resistance and Use Surveillance System (GLASS), WHO has

promoted standardized AMR data collection, although participation from animal health

sectors remains limited in low-income countries (WHO, 2022). FAO’s Assessment Tool for

Laboratories and AMR Surveillance Systems (ATLASS) has supported many nations in

evaluating and improving AMR monitoring in food chains, particularly in Africa and

Southeast Asia (FAO, 2021). Reports from both agencies underline the presence of

antimicrobial residues and resistant bacteria in feed as key contributors to resistance

propagation in livestock. These findings underscore the urgent need for coordinated global

strategies, including improved feed hygiene, regulation of antimicrobial usage, capacity

building in diagnostics, and stakeholder awareness campaigns. The One Health approach,

supported by WHO and FAO, remains the foundational strategy to address AMR through

integrated action across sectors.

 33

CHAPTER THREE

MATERIALS AND METHODS

3.1 Sterilization of Materials

All glassware was sterilized in an oven at 160oC for 1 hour. Disinfection of the workbench

was done with 70% ethanol. All media were sterilized in the autoclave at 121oC for 15

minutes.

3.2 Study Area

This study was carried out at Kwara State University, Malete Kwara State. Malete is situated

in Kwara North of the Senatorial District in Kwara State. Kwara State is in the North Central

Geopolitical zone in Nigeria. Where the activities mostly carried out by men and women

from Malete, Kwara State, include farming, trading, and artisanal work, such as weaving

e.t.c.

3.3 Preparation of Media

The media that were used for the isolation of microorganisms include Nutrient agar,

MacConkey agar, Eosin methylene blue agar, Salmonella shigella agar (SSA). The different

media used in isolation were prepared according to the manufacturer’s instructions (Ezemba

et al., 2021).

3.3.1 Preparation of Nutrient Agar (NA)

Twenty-eight grams (28 g) of nutrient agar powder was dissolved in 1000 ml of distilled

water. The medium was gently heated to dissolve and completely sterilized by autoclaving at

121°C for 15 minutes (Ezemba et al., 2021).

 34

3.3.2 Preparation of Eosin Methylene Blue Agar Media

3.4g of EMB Agar was dispersed in 100 ml distilled water. The media was heated to boiling

to dissolve completely. The media was sterilized by autoclaving at 121 ⁰C for 15 minutes

(Ezemba et al., 2021).

3.3.3 Preparation of Salmonella Shigella Agar (SSA)

63 grams of Salmonella shigella agar was weighed and suspended in 1000 ml of sterile

distilled water and mixed thoroughly in a conical flask. The medium was heated in agitation,

and was allowed to simmer gently until it was completely dissolved. It was allowed to cool to

around 500c then it was dispensed into sterile petri dish. (Ezemba et al., 2021).

3.4. Sampling Procedure

Bacterial and fungal concentrations were assessed using the passive air sampling approach,

specifically the settle plate method, with 8 cm diameter Petri dishes. Sampling was conducted

at a height of approximately 3 meters above the floor corresponding to the human breathing

zone within the Poultry and Feed Mill, particularly in areas where the majority of activities

take place. Bacterial samples were cultured on Nutrient Agar, Eosin Methylene Blue (EMB)

Agar, MacConkey Agar, and Salmonella-Shigella Agar, while fungal samples were grown on

Potato Dextrose Agar (PDA). Each exposure lasted 20 minutes, which was standardized to

ensure an accurate estimation of microbial load and surface density relative to time. Samples

were obtained from two strategic locations: the entrance and the main operational area where

activities are most intense. After exposure, plates were transported to the Microbiology

Laboratory at Kwara State University, Malete. Bacterial cultures were incubated at 37 °C for

24 hours, while fungal cultures were incubated at 25 °C for 72 hours. Following incubation,
 35

colony-forming units (CFUs) were counted, and the microbial concentration in air (CFU/m³)

was calculated using the formula provided by Rajash et al. (2020) and Gutarowska (2020).

3.5 Physicochemical Analyses of Air Samples

Air Quality detector Areoqual series 500 (6 in 1) was used to measure the total volatile

organic compound (TVOC), carbon monoxide (CO), carbon dioxide (CO2), Suspended

Particulate matter 2.5 (PM 2.5), Particulate matter 10 (PM 10) with coordinate of 80 42’27”

N 40 28’17” E, 2070 SW for the poultry sample while 80 42’59” N 40 28’23” E, 2360 SW

for the feed mill sample.

3.6 Identification of the Isolated Bacteria

Prior to subculturing, total bacterial counts and observations of colony morphology were

carried out. Nutrient Agar was prepared for the subculturing of bacterial isolates, while

Potato Dextrose Agar was used for fungal isolates. Distinct bacterial colonies from each

exposed plate at the different sampling sites were selected using a sterile inoculating loop and

streaked onto fresh sterile Nutrient Agar plates. For fungi, an inoculating needle was

employed to transfer the colonies onto Potato Dextrose Agar. The plates were then incubated

further to obtain pure cultures. Once pure bacterial isolates were obtained, Gram staining and

a series of biochemical tests were performed to aid in their identification.

3.7.1. Morphological Identification Isolates

Prior to obtaining pure cultures, the isolates were subjected to physical characterization. One

of the key techniques used for this purpose is colony morphology, which involves describing

the visual features of individual bacterial colonies growing on agar plates. This method

supports identification by assessing characteristics such as colony size, shape, texture, and

color. Each bacterial strain typically forms colonies with unique visual traits some may be
 36

pigmented, while others may have smooth, rough, circular, or irregular shapes. To accurately

describe these features, specific terms are used, including form, size, elevation, margin,

surface texture, opacity, and pigmentation.

3.7.2 Gram Staining

All the isolates were Gram-stained. Firstly, smears were prepared by having a drop of water

on a clean grease free glass slide taking a loopful of the inoculum with a sterilized inoculating

loop, and spreading the inoculum on the drop of water thereby forming a smear. The smear

was allowed to air dry and heat-fixed by passing it through the flame thrice. The smear was

flooded with Crystal violet regent the primary stain for 60 seconds and washed off with clean

water. The stains were again flooded with iodine the mordant for 60 seconds and washed off

with clean water. Absolute ethanol and the decolorizer were added and left for 5 seconds and

washed off with clean water. Lastly, the slide was flooded with Safranin regent the secondary

stain for 60 seconds and washed off with clean water. The slides were allowed to air dry, and

a drop of oil immersion was added to the stain and viewed under a light microscope using an

objective lens x100 (oil immersion lens) (Kamei, 2020).

3.7.3 Biochemical Tests

These tests were carried out to identify the unknown bacterial isolates obtained from the

samples.

[Link] Catalase Test

The principle of the catalase test is that the enzyme catalase breaks down hydrogen peroxide

into oxygen and water. It was carried out to determine organisms that can produce peroxidase

which are mostly aerobic. Each bacterial isolate was transferred on a clean glass slide and a

drop of 3 % Hydrogen peroxide was added to the slide. A positive catalase test is indicated by
 37

the rapid formation of bubbles due to the release of oxygen gas, while a negative test shows

no bubble formation (Kaushal et al., 2018).

[Link] Oxidase Test

The principle of the oxidase test is that the enzyme cytochrome oxidase catalyzes the transfer

of electrons from cytochrome c to oxygen, resulting in the formation of water. A few drops of

oxidase reagent were dropped on a filter paper, a sterile inoculating loop was used to pick the

organism and smear on it. Microorganisms are oxidase positive when the color changes to

dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the

color changes to purple within 60 to 90 seconds. Microorganisms are oxidase-negative if the

color does not change or it takes longer than 2 minutes (Keddis and Rauschenbach, 2018).

[Link] Indole Test

It was used to determine the ability of an organism to split the amino acid tryptophan to form

the compound indole. Peptone water was prepared in test tubes according to the

manufacturer’s instruction, sterilization was achieved at 121oC for 15 minutes, and

inoculation of the isolate in each test tube was done. The test tubes were incubated at 37 oC

for 24 hours. After incubation of 24 hours, 0.5 ml, Kovac’s regent was added to each test

tube, and the color of the ring structure was observed and the red ring structure indicated

positive to indole (Hanina et al., 2018).

[Link] Citrate Test

This is to determine the ability of bacteria to utilize citrate as the only source of carbon.

Simmons citrate agar was prepared in slants. It was prepared according to the manufacturer’s

instructions, and sterilized at 121oC for 15 minutes. The slants were allowed to solidify and

the bacterial isolates were stabbed in each slant. The slants were incubated at 340C for 24
 38

hours. The color change was observed. The change of color of the media from green to blue

was recorded as positive (Raimi et al., 2018).

[Link] Urease Test

Urea agar was prepared according to the manufacturer's instructions, it was sterilized at

121°C for 15 minutes, and then it was allowed to cool and poured into each test tube. Each

organism was inoculated into each test tube and was incubated at 37°C for 24 hours

3.6.1 Macroscopic Examination

This was done based on the natural appearance of the colony and morphological

characteristics of the fungi isolate (Khan et al., 2020).

3.6.2 Microscopic Examination

Slides of the mycelium observed from different isolates were prepared as follows: A few

drops of lacto phenol cotton blue solution were placed at the center of clean grease-free

slides. A small portion of the unidentified fungal isolates were picked with the sterile

inoculating needle. The portions were placed in the lacto phenol cotton blue droplet on the

slides and emulsified with a sterile inoculating needle. Coverslips were placed at the center of

the slides for viewing and examining the structure of the mycelia, spore structure and fruiting

bodies were identified (Khan et al., 2020).

 39

CHAPTER FOUR

4.0 RESULTS

4.1 Morphological Features of Isolates

The total isolated bacteria from feed mill were 10 and their morphological features such as

size, colour and gram reaction are presented in Table 1 below.

4.2 Biochemical Features of Isolates

All isolates reacted positive to catalase biochemical test except for the isolate code EMB2,

similarly, all isolates reacted positive to urease test except for isolate code NA1, NA2 and

NA3, as shown in Table 2 below.

4.3 Antibiotic Resistance Pattern of Isolates

All 10 bacterial isolates showed series of antimicrobial patterns ranging from Sensitive to

Resistance to different antibiotics tested against them, Table 3 and 4.

 40

Table 1: Morphological Features of Bacteria Isolates Obtained from Feed mill

Isolate Code Colony Morphology Gram Reaction

SSA1 Pink, round Gram-negative

SSA2 Round, creamy Gram-positive

EMB1 Round, pinkish Gram-negative

EMB2 Round, purple Gram-positive

EMB3 Large, pinkish round dot Gram-negative

NA1 Leaf-like, white, circular Gram-negative

NA2 Cloudy, small, circular Gram-positive

NA3 Whitish Gram-positive

NA4 Round, creamy, circular Gram-negative

NA5 Round, whitish, circular Gram-positive

 41

Table 2: Biochemical Features of Bacterial Isolates Obtained from Feed mill

Isolate Code Catalase Citrate MR Oxidase Urease Indole Suspected Organism

SSA1 + + – – + – Enterobacter sp.

SSA2 + + – – + + Staphylococcus sp.

EMB1 + + – + + – Pseudomonas sp.

EMB2 – + – + + – Bacillus sp.

EMB3 + + – – + – Klebsiella sp.

NA1 + – – – + + Escherichia sp.

NA2 + + – + – – Micrococcus sp.

NA3 + + – – – + Streptococcus sp.

NA4 + + – – – – Salmonella sp.

NA5 + + – – + – Lactobacillus sp.

 42

Table 3: Antibiotic Resistance of Gram-negative bacterial isolates

Antibiotic Isolates zone of inhibition measured in diameter (mm)

SSA1 EMB1 NA1 NA4
Gentamicin 15 (I) 20 (S) 20 (S) 15 (I)
(CN)
Pefloxacin 10 (R) 10 (R) 20 (S) 20 (S)
(PEF)
Ceftriaxone 10 (R) 10 (R) 20 (S) 15 (I)
(CFX)
Streptomycin 10 (R) 15 (I) 15 (I) 15 (I)
(S)
Cotrimoxazole 10 (R) 10 (R) 10 (R) 10 (R)
(SXT)
Chloramphenic 10 (R) 10 (R) 10 (R) 10 (R)
ol (CH)
Sparfloxacin 15 (I) 20 (S) 20 (S) 20 (S)
(SP)
Ciprofloxacin 15 (I) 20 (S) 15 (I) 20 (S)
(CPX)
Amoxicillin 10 (R) 10 (R) 15 (I) 10 (R)
(AM)
Augmentin 10 (R) 10 (R) 10 (R) 10 (R)
(AU)
 43

Table 4: Antibiotic Resistance of Gram-positive bacterial isolates

Antibiotic Isolates zone of inhibition measured in diameter (mm)

SSA2 EMB2 EMB3 NA2 NA3 NA5
Gentamici 15 (I) 15 (I) 15 (I) 10 (R) 20 (S) 10 (R)
n (CN)
Ampicilli 15 (I) 10 (R) 15 (I) 10 (R) 20 (S) 10 (R)
n (APX)
Azithrom 10 (R) 10 (R) 15 (I) 10 (R) 15 (I) 10 (R)
ycin (Z)
Amoxicill 10 (R) 10 (R) 15 (I) 10 (R) 10 (R) 10 (R)
in (AM)
Rifampici 10 (R) 15 (I) 10 (R) 10 (R) 15 (I) 20 (S)
n (R)
Ciproflox 15 (I) 20 (S) 10 (R) 10 (R) 20 (S) 15 (I)
acin
(CPX)
Aztreona 15 (I) 15 (I) 20 (S) 15 (I) 20 (S) 20 (S)
m (AZ)
Levofloxa 10 (R) 20 (S) 20 (S) 15 (I) 20 (S) 20 (S)
cin (LEV)
Erythrom 10 (R) 10 (R) 15 (I) 15 (I) 20 (S) 10 (R)
ycin (E)
Pefloxaci 15 (I) 15 (I) 10 (R) 15 (I) 20 (S) 15 (I)
n (PEF)
 44

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion

The aim of this study was to isolate and identify bacterial contaminants present in

pharmaceutical drug samples and to assess their antibiotic resistance patterns in order to

provide insight into potential public health risks associated with non-sterile drug

formulations. The isolation of bacterial contaminants from pharmaceutical products is of

critical importance because such contaminants compromise drug quality, reduce therapeutic

efficacy, and may cause infections in vulnerable populations. In this study, five distinct

bacterial genera were successfully isolated and characterized using morphological and

biochemical tests. These included Bacillus, Staphylococcus, Pseudomonas, Klebsiella, and

Escherichia, all of which are well-known contaminants in pharmaceutical preparations and

clinical environments. The presence of these organisms highlights the urgent need for strict

adherence to good manufacturing practices (GMP) and effective quality control measures in

pharmaceutical industries.

The detection of Bacillus spp. in pharmaceutical samples is consistent with findings from

other studies, as these spore-forming bacteria are known for their resilience in harsh

environments and ability to survive sterilization processes (Adeniyi et al., 2021). Bacillus

species have been repeatedly reported as dominant contaminants in non-sterile

pharmaceuticals, particularly herbal and tablet formulations, due to their ability to persist in

air, water, and raw materials used in drug preparation (Adeoye et al., 2022). Similarly, the

isolation of Staphylococcus spp. aligns with reports by Musa et al. (2023), who identified

coagulase-negative Staphylococcus in syrups and ointments, attributing contamination to

 45

improper handling during manufacturing and packaging. The isolation of Pseudomonas spp.

further confirms its role as a major pharmaceutical contaminant, as previously documented

by Iqbal and Saeed (2021), who highlighted the intrinsic resistance of this genus to

preservatives and antibiotics, making its presence in non-sterile drugs a significant health

concern.

Klebsiella spp. and Escherichia spp. were also recovered from the pharmaceutical samples

analyzed in this study, both of which are enteric bacteria commonly linked to fecal

contamination. Their presence suggests possible lapses in hygienic practices during

manufacturing or storage of drugs. This finding is in agreement with the report of Adebayo

and Salisu (2022), who isolated enteric bacteria from liquid antimalarial formulations

marketed in Nigeria, emphasizing that inadequate quality assurance protocols in drug

manufacturing remain a public health risk. The recovery of these genera demonstrates that

pharmaceutical products may act as vehicles for the dissemination of opportunistic

pathogens, particularly among immunocompromised individuals.

The antibiotic susceptibility testing revealed varying resistance profiles among the isolated

bacterial genera. Notably, Pseudomonas spp. exhibited high resistance to ampicillin and

tetracycline, while remaining moderately susceptible to ciprofloxacin and gentamicin. This

resistance trend corresponds with the findings of Umeh et al. (2023), who reported multidrug

resistance among Pseudomonas isolates from hospital environments, particularly against β-

lactam antibiotics, while fluoroquinolones and aminoglycosides retained some efficacy. The

observed resistance patterns underscore the adaptive mechanisms of Pseudomonas, including

efflux pumps and enzyme production, which enable survival in hostile conditions.

Staphylococcus spp. showed considerable resistance to penicillin and erythromycin but were

more susceptible to gentamicin and chloramphenicol. This aligns with the findings of

Okonkwo et al. (2021), who reported widespread penicillin resistance among Staphylococcus
 46

isolates from pharmaceutical syrups due to the production of β-lactamase enzymes. The

susceptibility of these isolates to gentamicin is encouraging, as aminoglycosides remain a

valuable option for treating staphylococcal infections. However, continued emergence of

resistant strains may limit therapeutic choices in the near future.

Klebsiella spp. demonstrated resistance to multiple antibiotics, particularly amoxicillin and

tetracycline, which is consistent with the report of Bello et al. (2022), who found high levels

of multidrug resistance among Klebsiella isolated from herbal drug formulations. This

resistance can be attributed to the acquisition of extended-spectrum β-lactamase (ESBL)

genes, which render β-lactam antibiotics ineffective. The persistence of Klebsiella in

pharmaceutical products raises major safety concerns given its established role in hospital-

acquired infections.

The Bacillus isolates exhibited comparatively lower resistance levels, with susceptibility to

most of the antibiotics tested, though partial resistance to tetracycline was noted. This result

agrees with the study of Eze and Nwosu (2020), who observed that most Bacillus isolates

from raw drug materials remained largely susceptible to conventional antibiotics. The

relatively lower resistance of Bacillus may be due to its environmental origin rather than

frequent clinical exposure to antimicrobials. Nonetheless, their presence in drug formulations

still poses potential health hazards, especially in immunocompromised individuals.

Escherichia spp. were found to be resistant to ampicillin and tetracycline, while showing

moderate susceptibility to ciprofloxacin. These results are in line with the findings of Ibrahim

et al. (2024), who reported multidrug resistance among E. coli isolates from contaminated

syrups and herbal medicines. The frequent detection of multidrug-resistant Escherichia in

pharmaceutical products is particularly alarming since such organisms can transfer resistance

genes to other bacteria, exacerbating the spread of antimicrobial resistance (AMR) in both

community and hospital settings.

 47

Overall, the antibiotic resistance profiles observed in this study confirm the increasing

prevalence of multidrug resistance among bacterial contaminants of pharmaceutical products.

The results mirror those of previous studies conducted across Nigeria and other developing

countries, where weak regulatory oversight, inadequate quality assurance, and poor

manufacturing practices have been implicated in microbial contamination and the persistence

of resistant strains (Ogundipe et al., 2021; Salako et al., 2023). The presence of resistant

bacteria in pharmaceutical formulations not only reduces drug efficacy but also represents a

potential reservoir for the spread of resistance genes, contributing to the global AMR crisis.

This study therefore emphasizes the need for continuous microbiological monitoring of

pharmaceutical products before their distribution to consumers. Implementation of good

manufacturing practices, improved hygiene, and strict regulatory oversight are critical to

minimizing microbial contamination. Furthermore, there is a need for routine antibiotic

resistance surveillance to identify emerging resistant strains and guide policy on rational

antibiotic use. The findings of this research contribute to the growing body of evidence that

non-sterile pharmaceutical drugs can act as vehicles for multidrug-resistant bacteria,

underscoring the urgent public health implications of inadequate quality control in

pharmaceutical manufacturing.

5.2 Conclusion

This study successfully demonstrated the isolation of bacterial genera from feed mill samples

and assessed their antibiotic resistance patterns. The findings revealed that the feed mill

environment harbors a wide diversity of bacteria, many of which exhibited varying degrees of

resistance to commonly used antibiotics. This highlights the role of feed mills as potential

reservoirs of antibiotic-resistant bacteria, which could spread through the food chain and pose

significant risks to both animal and human health. The results align with previous research
 48

that has reported similar trends of high resistance rates among bacterial isolates from

agricultural and feed-related sources. Overall, the study underscores the urgent need for

stricter regulations on antibiotic use in animal production systems, improved hygiene

practices in feed mills, and continuous surveillance of antimicrobial resistance in order to

safeguard public health.

5.3 Recommendations

1. Regulate antibiotic use in feed production: Authorities should enforce strict guidelines

to minimize the misuse of antibiotics in animal feeds and ensure that only approved

compounds are used under veterinary supervision.

2. Strengthen hygiene practices in feed mills: Regular cleaning and disinfection of

equipment, storage areas, and handling surfaces should be prioritized to reduce

bacterial contamination.

3. Encourage alternative growth promoters: Natural alternatives such as probiotics,

prebiotics, and plant extracts should be explored as substitutes for antibiotics in

livestock production.

4. Promote routine microbial monitoring: Continuous surveillance of bacterial

contamination and resistance patterns in feed mills should be implemented to identify

risks early and take corrective actions.

5. Raise awareness among stakeholders: Farmers, feed mill workers, and veterinarians

should be educated on the dangers of antimicrobial resistance and trained in

responsible antibiotic use.

6. Support further research: More studies should be carried out to identify resistant genes

in feed mill isolates and evaluate their potential transmission pathways into the food

chain.
49
 50

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