CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

In recent years, the availability and consumption of sachet water, commonly referred to as “pure

water,” have surged dramatically in many urban areas of Nigeria, including Abakaliki, Ebonyi

State. This increase in demand is driven by the growing population and the need for accessible

and affordable potable water. However, despite its convenience, there are growing concerns

regarding the safety and quality of sachet water, which is essential for public health (Onyeneke

et al., 2020). This study aims to evaluate the physico-chemical and microbial qualities of selected

sachet water brands sold in Abakaliki to address these concerns.

The physico-chemical properties of water are critical indicators of its quality and suitability for

consumption. These properties include parameters such as pH, turbidity, total dissolved solids

(TDS), hardness, and concentrations of various ions. pH levels, for instance, are important

because water that is too acidic or alkaline can have adverse effects on health and taste. Turbidity

measures the cloudiness of water, which can be an indicator of particulate contamination. Total

dissolved solids reflect the presence of inorganic and organic substances that can affect the

water's taste and safety. Hardness, which is attributed to the presence of calcium and magnesium

ions, can affect both the health and taste of water (Jaffar et al., 2020).

Microbial contamination is another significant concern in sachet water. The presence of

pathogens such as bacteria, viruses, and protozoa can pose serious health risks, including

gastrointestinal infections and other waterborne diseases (Nwadike et al., 2024). Microbial

quality is typically assessed by testing for indicators such as total coliforms, Escherichia coli (E.
coli), and fecal coliforms. These microorganisms are commonly used as indicators of fecal

contamination and the overall sanitary quality of the water. The presence of such contaminants

suggests inadequate treatment or handling processes, which could endanger consumer health

(Wen et al., 2020).

In places like Abakaliki, where sachet water is a prevalent choice for drinking, ensuring that

these products meet acceptable standards is of paramount importance. The sachet water industry,

though regulated, often faces challenges related to quality control and adherence to safety

standards. Many sachet water vendors operate with limited resources, which can impact the

quality of the water produced. Additionally, improper handling, storage, and distribution

practices can further compromise the safety of sachet water (Ighalo and Adeniyi, 2020).

In conclusion, this study seeks to fill a gap in knowledge by providing a comprehensive

assessment of both the physico-chemical and microbial qualities of sachet water available in

Abakaliki. By analyzing these factors, the research aims to identify any potential quality issues

and offer recommendations for improving water safety and public health outcomes.

1.2 Justification of the Study

The widespread consumption of sachet water in Abakaliki, Ebonyi State, raises significant

concerns about its quality and safety. While sachet water is often viewed as an affordable and

convenient source of drinking water, reports of contamination have emerged, highlighting

potential health risks to consumers. Poor regulation, inadequate treatment processes, and

improper handling of sachet water by vendors can lead to unsafe levels of microbial

contamination, including the presence of harmful pathogens such as coliform bacteria, which are

known to cause waterborne diseases.

Furthermore, the physico-chemical qualities of sachet water, such as pH, turbidity, and total

dissolved solids (TDS), may fall outside the acceptable standards set by health authorities,

compromising both the safety and palatability of the water. The absence of regular monitoring

and enforcement of quality standards in the sachet water industry adds to the problem,

potentially exposing the population to harmful substances and contaminants.

Given these risks, there is an urgent need for a thorough screening of the physico-chemical and

microbial qualities of sachet water sold in Abakaliki. Without adequate knowledge of the quality

of water being consumed, public health remains at risk, with vulnerable populations, such as

children and the elderly, being particularly susceptible to waterborne diseases and other health

issues. This study seeks to address these concerns by evaluating the safety and quality of sachet

water, thereby informing regulatory bodies, producers, and consumers about the potential risks

associated with its consumption.

1.3 Aim and Objectives of the Study

The aim of this study is to evaluate the physico-chemical and microbial qualities of selected

sachet water sold in Abakaliki, Ebonyi State.

The specific objectives for the study include:

1. To determine the physicochemical properties of the sachet water samples.

2. To determine the concentration of metals in the samples

3. To isolate E. coli from water samples

4. To confirm the identity of the isolate

5. To perform antibiotics susceptibility testing of the E. coli isolate

6. To determine MARP and MARI

1.4 Literature Review

1.4.1 Overview of Water

Water is one of the most essential resources on Earth, vital to all forms of life and ecosystems. It

is a chemical compound composed of two hydrogen atoms and one oxygen atom (H₂O), existing

in liquid, solid, and gaseous states. Covering about 71% of the Earth's surface, water is found in

oceans, rivers, lakes, glaciers, underground reservoirs, and the atmosphere. Despite its

abundance, only about 2.5% of Earth's water is freshwater, suitable for drinking, with the vast

majority being saline water in oceans (Mocek-Płóciniak and Skowrońska, 2021).

1.4.1.1 Types of Water

1. Surface Water: Surface water is found on the Earth's surface in natural bodies such as rivers,

lakes, ponds, and oceans. It is a primary source of freshwater for human consumption,

agriculture, and industry. However, surface water is highly vulnerable to pollution from

industrial waste, agricultural runoff, and domestic sewage, which may introduce contaminants

like chemicals, heavy metals, and pathogens (Singh, 2024).

2. Groundwater: Groundwater is water located beneath the Earth's surface in aquifers, which are

layers of rock or soil that store water. It is a major source of freshwater, especially in areas where

surface water is scarce. Groundwater is often considered cleaner than surface water since it

undergoes natural filtration through soil layers. However, it can still become contaminated from

agricultural chemicals, industrial waste, and poor sanitation practices (Tiwari and Pal, 2021).
3. Rainwater: Rainwater is water that falls from the atmosphere as precipitation, including rain,

snow, hail, and sleet. It is a renewable source of freshwater and can be harvested for drinking,

agriculture, and domestic use. In areas with limited access to clean water, rainwater harvesting is

a sustainable method to meet water needs (Mishra, 2023).

4. Spring Water: Spring water originates from underground sources that naturally flow to the

Earth's surface. It is often considered pure because it is naturally filtered as it moves through

rock formations. Spring water is commonly bottled and sold as drinking water. However, like

groundwater, it can still be contaminated by surrounding environmental factors, so testing is

required to ensure its safety (Jung et al., 2024).

5. Saline Water (Saltwater): Saline water, found primarily in oceans and seas, contains high

concentrations of dissolved salts, particularly sodium chloride (NaCl). Saltwater makes up about

97% of the Earth's water and is not suitable for drinking or irrigation due to its salinity. However,

it is essential for marine life and ecosystems (Velmurugan et al., 2020).

6. Wastewater: Wastewater is water that has been used in households, industries, or agriculture

and contains contaminants from human activities. It includes sewage, industrial effluents, and

agricultural runoff. Wastewater treatment plants are designed to remove contaminants and purify

the water for safe discharge into the environment or for reuse in various non-drinking

applications (Karri et al., 2021).

1.4.1.2 Wastewater and It’s Properties

Wastewater refers to water that has been used in various activities such as domestic, industrial,

and agricultural processes and has become contaminated with organic and inorganic substances.

Once wastewater is generated, it can no longer be used directly for drinking or other purposes
without treatment, as it typically contains a range of pollutants that pose environmental and

health risks (Karri et al., 2021)

Sources of Wastewater

1. Domestic Wastewater (Sewage): This includes water from households, generated by

activities such as bathing, cooking, laundry, and sewage.

2. Industrial Wastewater: Water used in manufacturing processes, chemical production, mining,

and energy generation. Industrial wastewater may contain hazardous chemicals, heavy

metals, oils, solvents, and other toxic substances that can contaminate water sources.

3. Agricultural Wastewater: Water from agricultural activities such as irrigation, animal

husbandry, and pesticide application. This type of wastewater often contains fertilizers,

pesticides, herbicides, and organic materials like animal waste.

4. Stormwater Runoff: Water that flows over surfaces during rainfall events, collecting

pollutants from urban areas, roads, and industrial sites. Stormwater can carry sediments,

oils, chemicals, and other contaminants into water bodies.

Qualities of Wastewater

1. Physical Qualities of Wastewater:

i. Turbidity: Wastewater often contains suspended solids that contribute to its turbidity or

cloudiness. These solids can include organic matter, silt, debris, and other particles. High

turbidity indicates poor water clarity and can affect aquatic life by blocking sunlight and

reducing oxygen levels (Owodunni and Ismail, 2021).

 ii. Color: Wastewater may have varying colors depending on its origin. Domestic

wastewater may appear grey or brown, while industrial wastewater may have distinct

colors due to dyes, chemicals, or other pollutants (Manasa and Mehta, 2020).

iii. Temperature: Wastewater, especially from industrial processes, may be discharged at

elevated temperatures, which can disrupt aquatic ecosystems by affecting dissolved

oxygen levels and altering habitat conditions for aquatic organisms (Hamdhani et al.,

2020).

2. Chemical Qualities of Wastewater:

i. pH: The acidity or alkalinity of wastewater is measured by its pH. A pH outside the

neutral range (6.5–8.5) can indicate contamination by acidic or basic substances.

Industrial wastewater may have extreme pH levels due to chemical discharges (Wokoma

and Edori, 2020).

ii. Organic Content (Biochemical Oxygen Demand - BOD): Wastewater contains organic

matter such as proteins, fats, carbohydrates, and oils. The BOD measures the amount of

oxygen that microorganisms need to break down these organic substances. High BOD

levels indicate high organic pollution, which can lead to oxygen depletion in water

bodies, harming aquatic life (Manasa and Mehta, 2020).

iii. Chemical Oxygen Demand (COD): COD measures the total amount of oxygen required

to oxidize both organic and inorganic compounds in wastewater. It provides an estimate

of the water's pollution level (Aguilar-Torrejón et al., 2023).

iv. Nutrients (Nitrogen and Phosphorus): Wastewater often contains high levels of

nutrients, particularly nitrogen and phosphorus, from fertilizers, detergents, and organic
 matter. Excessive nutrients can lead to eutrophication, a process that results in the

overgrowth of algae in water bodies, reducing oxygen levels and harming aquatic life

(Weerakoon et al., 2023).

v. Heavy Metals: Industrial wastewater may contain heavy metals such as lead, mercury,

cadmium, and arsenic, which are toxic to humans and animals, even at low

concentrations. These metals can accumulate in the food chain, leading to long-term

health and environmental issues (Dahiya, 2022).

vi. Salts and TDS (Total Dissolved Solids): Wastewater, especially from agricultural and

industrial sources, may have high levels of dissolved salts, which can impact soil fertility

and water quality. TDS measures the concentration of dissolved ions such as calcium,

magnesium, sodium, and chloride (Younas and Younas, 2022).

3. Biological Qualities of Wastewater:

i. Pathogens: Wastewater contains a wide range of pathogens, including bacteria, viruses,

protozoa, and helminths (parasitic worms). These microorganisms, especially in

blackwater, pose a serious public health risk if not treated properly. Common pathogens

found in wastewater include Escherichia coli (E. coli), Salmonella, Vibrio cholerae

(causes cholera), and rotavirus (Bej et al., 2023).

ii. Coliform Bacteria: The presence of coliform bacteria, particularly fecal coliforms,

indicates fecal contamination and the potential presence of harmful pathogens (Kumar et

al., 2021).

iii. Algae and Fungi: Wastewater can also support the growth of algae and fungi, especially

when nutrients like nitrogen and phosphorus are abundant. While some of these
 microorganisms are harmless, others can produce toxins that are harmful to both humans

and aquatic life (Wu et al., 2023).

1.4.2 Overview of Enterobacteriaceae

Enterobacteriaceae is a large family of Gram-negative bacteria that includes many genera of

medical and ecological importance. These bacteria are commonly found in the intestines of

humans and animals, as well as in soil, water, and plants. The family includes both harmless

symbionts, which are part of the normal gut flora, and pathogenic species that can cause a wide

range of infections, including gastrointestinal diseases, urinary tract infections, and systemic

infections (Janda and Abbott, 2021).

1.4.2.1 Characteristics of Enterobacteriaceae

1. Gram-negative Rods: Enterobacteriaceae are rod-shaped bacteria that stain pink in Gram

staining due to their thin peptidoglycan cell wall surrounded by an outer membrane.

2. Facultative Anaerobes: These bacteria can grow in both the presence and absence of

oxygen, giving them the ability to survive in a variety of environments.

3. Fermentation: Many members of Enterobacteriaceae can ferment glucose and other

carbohydrates, producing acid and sometimes gas. This ability is used as a key feature in

biochemical identification.

4. Oxidase-negative: Most members of Enterobacteriaceae lack the enzyme cytochrome c

oxidase, which is used in the identification of bacterial species.

5. Nitrate Reduction: They can reduce nitrate to nitrite, which is another biochemical trait

used for differentiation.

Genera and Species of Medical Importance

1. Escherichia coli (E. coli): While many strains of E. coli are harmless and are part of the

normal gut flora, certain pathogenic strains can cause serious infections. Enterotoxigenic E.

coli (ETEC) causes traveler’s diarrhea, while enterohemorrhagic E. coli (EHEC), can cause

severe foodborne illness and hemolytic-uremic syndrome (HUS) (Cabrera-Sosa and Ochoa,

2020).

2. Salmonella: Salmonella species, particularly Salmonella enterica, are known for causing

foodborne illnesses. Salmonella typhi and Salmonella paratyphi cause typhoid and

paratyphoid fever, respectively, which are life-threatening systemic infections (Ayuti et al.,

2024).

3. Klebsiella pneumoniae: Klebsiella species are significant causes of hospital-acquired

infections, particularly in immunocompromised patients. Klebsiella pneumoniae can cause

pneumonia, bloodstream infections, and urinary tract infections (Alharbi et al., 2023).

4. Shigella: Shigella species are responsible for bacillary dysentery or shigellosis, a severe

diarrheal disease that is spread through contaminated food and water. The infection is

particularly dangerous in children and in areas with poor sanitation (Ahamed and Giria,

2021).

5. Enterobacter: Enterobacter species, like Enterobacter cloacae, are opportunistic pathogens

that can cause infections in hospitalized patients, particularly in those with weakened

immune systems. These bacteria can lead to respiratory, urinary tract, and bloodstream

infections (Anju et al., 2020).

6. Proteus: Proteus mirabilis is a common cause of urinary tract infections, particularly in

individuals with long-term catheterization. It is known for its ability to produce urease, which
 breaks down urea into ammonia, leading to the formation of kidney stones (Yuan et al.,

2021).

7. Serratia: Serratia marcescens is an opportunistic pathogen often associated with hospital-

acquired infections. It can cause respiratory, urinary, and wound infections, particularly in

immunocompromised individuals (Sheykhsaran et al., 2022).

Pathogenicity and Virulence Factors

1. Adhesion Factors: Many Enterobacteriaceae possess fimbriae or pili, which allow them to

adhere to host tissues and establish infections.

2. Toxins: Several species produce toxins that contribute to their pathogenicity. For example, E.

coli produces shiga toxins in the case of enterohemorrhagic strains.

3. Capsule Formation: Some species, such as Klebsiella pneumoniae, produce a thick

polysaccharide capsule that protects them from phagocytosis and enhances their ability to

cause infections.

4. Antibiotic Resistance: The spread of antibiotic resistance, especially among Klebsiella,

Enterobacter, and E. coli, poses a significant challenge in clinical settings. Many

Enterobacteriaceae produce β-lactamases, including extended-spectrum β-lactamases

(ESBLs) and carbapenemases, making them resistant to broad-spectrum antibiotics (De

Angelis et al., 2020).

1.4.3 Overview of Escheichia coli

Escherichia coli (E. coli) is a Gram-negative, rod-shaped bacterium that belongs to the family

Enterobacteriaceae. It is a highly diverse species, found naturally in the intestines of humans and

animals, where it plays an essential role in digestion. While most strains of E. coli are harmless
and part of the normal gut flora, some strains are pathogenic and can cause serious diseases in

humans, ranging from mild gastroenteritis to severe, life-threatening infections (Malabadi et al.,

2024).

1.4.3.1 General Characteristics of E. coli

1. Gram-negative: E. coli stains pink in Gram staining due to its thin peptidoglycan layer and

outer membrane structure.

2. Facultative anaerobe: It can grow in both the presence and absence of oxygen, making it

adaptable to a wide range of environments.

3. Motility: Some E. coli strains are motile, possessing peritrichous flagella that allow them to

move.

4. Lactose Fermentation: E. coli can ferment lactose, producing acid and gas, which is a key

characteristic used in the identification of the bacterium.

5. Oxidase-negative: It does not produce cytochrome c oxidase, which differentiates it from

other bacteria.

1.4.3.2 Types of E. coli

1. Enterotoxigenic E. coli (ETEC): It is a common cause of traveler’s diarrhea, especially in

developing countries and also produces toxins (heat-labile and heat-stable enterotoxins) that

lead to watery diarrhea (Cabrera-Sosa and Ochoa, 2020).

2. Enteropathogenic E. coli (EPEC): It causes diarrhea, particularly in infants in developing

countries (Cabrera-Sosa and Ochoa, 2020).

3. Enterohemorrhagic E. coli (EHEC): This includes strains like E. coli O157:H7, which can

cause severe foodborne illness. Also, it also produces shiga toxins, leading to bloody
 diarrhea and, in severe cases, hemolytic uremic syndrome (HUS), which can result in kidney

failure (Werner et al., 2024).

4. Enteroinvasive E. coli (EIEC): It invades and multiplies within intestinal epithelial cells,

causing inflammatory diarrhea similar to that caused by Shigella. It is characterized by

fever, abdominal cramps, and dysentery-like symptoms (Makarova, 2023).

5. Enteroaggregative E. coli (EAEC): This type of E. coli adheres to the intestinal mucosa in

a characteristic stacked-brick pattern and causes persistent diarrhea, particularly in children

and immunocompromised individuals (Cabrera-Sosa and Ochoa, 2020).

1.4.3.3 Pathogenesis and Virulence Factors

Pathogenic strains of E. coli have evolved various mechanisms to cause disease. These include:

i. Fimbriae (Pili): Many strains of E. coli possess fimbriae that allow them to adhere to host

cells, particularly in the gastrointestinal or urinary tracts. This adhesion is crucial for

colonization and infection (Pakbin et al., 2021).

ii. Toxins: Some strains produce powerful toxins that damage host cells. For example, shiga

toxins produced by EHEC can lead to cell death, bloody diarrhea, and systemic

complications such as HUS (Pakbin et al., 2021)

iii. Invasions: Some pathogenic E. coli strains can invade host cells and tissues, causing

localized infections or systemic disease (Pakbin et al., 2021).

iv. Capsule and Surface Proteins: These protect E. coli from phagocytosis and immune

responses, allowing the bacteria to survive in the host for longer periods (Pakbin et al.,

2021).

v. Hemolysins: Some strains produce hemolysins that lyse red blood cells, contributing to

tissue damage and inflammation (Pakbin et al., 2021).

 1.4.3.4 Transmission of E. coli

i. Fecal-Oral Route: Most E. coli infections are transmitted through the fecal-oral route,

meaning the bacteria are ingested through contaminated food, water, or contact with fecal

matter. Poor hygiene, inadequate sanitation, and improper food handling can facilitate the

spread (Gourama, 2020).

ii. Foodborne Illness: EHEC, in particular, is often spread through undercooked meat

(especially beef), unwashed vegetables, unpasteurized milk, and contaminated water

(Cappelier, 2022).

1.4.3.5 Diagnosis of E. coli infection

The diagnosis of E. coli infections depends on the type of infection. For gastrointestinal

infections, stool samples are tested for the presence of pathogenic strains (Negrut et al., 2020).

Urinary tract infections are diagnosed by culturing urine samples, while blood cultures can detect

E. coli in cases of septicemia. Molecular techniques like polymerase chain reaction (PCR) can

detect specific virulence genes to identify pathogenic strains, particularly during outbreaks

(Loderstädt et al., 2021).

1.4.4 Overview of Antibiotics

Antibiotics are powerful chemical substances used to treat infections caused by bacteria. They

work by either killing bacteria (bactericidal) or inhibiting their growth (bacteriostatic). The

discovery of antibiotics, starting with penicillin by Alexander Fleming in 1928, revolutionized

medicine by providing a way to treat bacterial infections effectively. Today, antibiotics are

essential tools in treating a wide range of infections, including pneumonia, tuberculosis, sepsis,

and urinary tract infections, among others (Chhabra et al., 2024).

However, the overuse and misuse of antibiotics have led to the emergence of antibiotic-resistant

bacteria, posing a global health threat. Antibiotics are classified into various groups based on

their chemical structure, mechanism of action, and the spectrum of bacteria they target (Uddin et

al., 2021).

1.4.4.1 Mechanism of Action of Antibiotics

1. Inhibitors of Cell Wall Synthesis: These antibiotics target the bacterial cell wall, which is

absent in human cells, making them selective for bacterial infections. They interfere with the

synthesis of peptidoglycan, a crucial component of the bacterial cell wall, resulting in

bacterial lysis (Shan et al., 2020). Some antibiotics that use this mechanism of action

include:

i. Penicillins: First discovered antibiotic class, effective against Gram-positive bacteria.

Examples include Penicillin G, Amoxicillin, and Ampicillin.

ii. Cephalosporins: Similar to penicillins but with a broader spectrum. Examples include

Ceftriaxone, Cefazolin, and Cefuroxime.

iii. Carbapenems: Very broad-spectrum antibiotics, effective against multi-drug-resistant

bacteria. Examples are Imipenem and Meropenem.

iv. Monobactams: Active primarily against Gram-negative bacteria. An example of

monobactam is Aztreonam.

v. Glycopeptides: Inhibit cell wall synthesis in Gram-positive bacteria.

2. Protein Synthesis Inhibitors: These antibiotics target the bacterial ribosome, which differs

in structure from human ribosomes, preventing bacterial protein synthesis (Anandabaskar,

2021).
 i. Aminoglycosides: Bind to the 30S subunit of bacterial ribosomes, causing misreading of

mRNA. Examples: Gentamicin, Streptomycin, Amikacin.

ii. Tetracyclines: Bind to the 30S subunit, blocking the attachment of tRNA to the ribosome.

Examples: Doxycycline, Tetracycline, Minocycline.

iii. Macrolides: Bind to the 50S subunit, inhibiting translocation of ribosomes along mRNA.

Examples: Erythromycin, Azithromycin, Clarithromycin.

iv. Lincosamides: Similar mechanism to macrolides, effective against Gram-positive

bacteria. Example: Clindamycin.

3. Nucleic Acid Synthesis Inhibitors: These antibiotics interfere with the synthesis of

bacterial DNA or RNA, inhibiting bacterial replication (Bhattacharjee, 2022).

i. Quinolones/Fluoroquinolones: Inhibit DNA gyrase and topoisomerase IV, enzymes

required for bacterial DNA replication. Examples: Ciprofloxacin, Levofloxacin,

Moxifloxacin.

ii. Rifamycins: Inhibit bacterial RNA polymerase, blocking RNA synthesis. Example:

Rifampicin (commonly used for tuberculosis).

iii. Metronidazole: Disrupts bacterial DNA by causing strand breaks, effective against

anaerobic bacteria and some protozoa.

4. Inhibitors of Metabolic Pathways: These antibiotics disrupt key metabolic processes, such

as folic acid synthesis, which is essential for bacterial DNA production (Roemhild et al.,

2022).

i. Sulfonamides: Inhibit dihydropteroate synthase, blocking folic acid synthesis. Example:

Sulfamethoxazole.
 ii. Trimethoprim: Inhibits dihydrofolate reductase, preventing the conversion of

dihydrofolate to tetrahydrofolate in the folic acid pathway.

5. Disruption of Cell Membranes: These antibiotics damage the bacterial cell membrane,

leading to leakage of cell contents and cell death (Benfield and Henriques, 2020).

i. Polymyxins: Bind to lipopolysaccharides in Gram-negative bacterial membranes, causing

disruption. Example: Polymyxin B, Colistin.

ii. Daptomycin: Disrupts the membrane potential in Gram-positive bacteria, leading to

bacterial death.

Classification of Antibiotics

1. Based on Spectrum of Activity

a. Narrow-Spectrum Antibiotics: These antibiotics are effective against a limited range of

bacteria, typically either Gram-positive or Gram-negative. They are preferred when the

causative bacteria are known to minimize the impact on normal flora. Examples: Penicillin G

(Gram-positive) and Aztreonam (Gram-negative) (Upmanyu and Malviya, 2020).

b. Broad-Spectrum Antibiotics: These antibiotics are effective against a wide range of

bacterial species, both Gram-positive and Gram-negative. Broad-spectrum antibiotics are

useful when the specific causative agent is unknown, but they can disrupt normal flora and

lead to resistance. Examples: Tetracycline, Ciprofloxacin, Amoxicillin-Clavulanic acid

(Upmanyu and Malviya, 2020)

2. Based on Origin
a. Natural Antibiotics: These antibiotics are derived from microorganisms such as fungi and

bacteria. For example, penicillin is produced by the fungus Penicillium. Example of natural

antibiotic is Penicillin (Schneider, 2021).

b. Semi-synthetic Antibiotics: These are chemically modified derivatives of natural antibiotics

to improve their efficacy, spectrum, or resistance to inactivation. Example: Amoxicillin (a

derivative of penicillin) (Qadri et al., 2022).

c. Synthetic Antibiotics: Completely man-made antibiotics, developed through chemical

processes, which often have no direct counterpart in nature. Example: Ciprofloxacin

(Grigalunas et al., 2022).

3. Based on Chemical Structure: Antibiotics can also be classified by their core chemical

structure, which defines their mode of action and the type of bacteria they affect (Li et al., 2021).

a. β-lactams: Penicillins, cephalosporins, carbapenems.

b. Aminoglycosides: Streptomycin, Gentamicin.

c. Macrolides: Erythromycin, Azithromycin.

d. Tetracyclines: Doxycycline, Minocycline.

e. Fluoroquinolones: Ciprofloxacin, Levofloxacin.

1.4.4.2 Antibiotic Resistance

Antibiotic resistance is a serious global health issue where bacteria evolve mechanisms to

withstand the effects of antibiotics that were previously effective against them. This makes

infections caused by these resistant bacteria more difficult to treat, leading to prolonged illness,

increased healthcare costs, and higher mortality rates. Antibiotic resistance has been accelerated
by the overuse and misuse of antibiotics in both humans and animals, as well as inadequate

infection control practices (Uddin et al., 2021).

Causes of Antibiotic Resistance

1. Overuse of Antibiotics: Excessive and unnecessary use of antibiotics, especially for viral

infections (e.g., colds, flu) where they are ineffective, contributes significantly to resistance

(Rahman et al., 2022).

2. Incomplete Treatment: Not completing prescribed antibiotic courses allows some bacteria

to survive, adapt, and become resistant (Andersson et al., 2020).

3. Use in Agriculture: Antibiotics are often used in livestock and agriculture to promote

growth and prevent diseases, which contributes to the development of resistant strains that

can be transmitted to humans through food or the environment (Vidovic and Vidovic, 2020).

4. Lack of New Antibiotics: The development of new antibiotics has slowed down in recent

decades, while resistance to existing drugs has increased, making it harder to find effective

treatments (Tarín-Pelló et al., 2022).

Mechanisms of Antibiotic Resistance

1. Enzymatic Degradation of Antibiotics: Some bacteria produce enzymes that chemically

modify or degrade antibiotics, rendering them ineffective. Some of these enzymes include:

a. β-lactamases: These enzymes break down β-lactam antibiotics (e.g., penicillins,

cephalosporins) by hydrolyzing the β-lactam ring, a crucial structure for antibiotic activity.
b. Extended-Spectrum β-Lactamases (ESBLs): These enzymes confer resistance to a wide

range of β-lactam antibiotics, including third-generation cephalosporins.

c. Carbapenemases: These enzymes degrade carbapenems, which are often considered last-

resort antibiotics for multi-drug-resistant infections.

2. Alteration of Target Sites: Bacteria can mutate or modify the target sites of antibiotics,

making it harder for the drugs to bind and exert their effects.

a. Penicillin-Binding Proteins (PBPs): In bacteria like Staphylococcus aureus and

Streptococcus pneumoniae, mutations in PBPs reduce the binding affinity of β-lactam

antibiotics.

b. Ribosomal Alterations: Some bacteria alter their ribosomal subunits, preventing antibiotics

like macrolides, tetracyclines, or aminoglycosides from binding and inhibiting protein

synthesis.

c. DNA Gyrase and Topoisomerase IV: Fluoroquinolones target these enzymes, and mutations

in their genes (e.g., gyrA, parC) can lead to resistance by reducing the drug’s ability to

inhibit DNA replication.

3. Efflux Pumps: Efflux pumps are protein systems in bacterial cell membranes that actively

pump out antibiotics, decreasing their intracellular concentration and preventing them from

reaching their targets.

a. Multidrug Resistance (MDR) Efflux Pumps: These pumps can remove a wide range of

antibiotics from the bacterial cell, contributing to resistance to multiple antibiotic classes,

such as tetracyclines, fluoroquinolones, and macrolides.

b. Specific Efflux Pumps: Some efflux pumps are specific to particular antibiotics, while others

can expel multiple classes of drugs, making bacteria resistant to a broader range of

treatments.

4. Reduced Permeability of the Bacterial Cell Membrane: Bacteria can alter their cell wall

or membrane structure to reduce the entry of antibiotics into the cell.

a. Porin Mutations: Gram-negative bacteria have porin channels in their outer membrane that

allow small molecules, including antibiotics, to enter. Mutations that reduce the size or

number of these porins can prevent antibiotics like β-lactams and aminoglycosides from

entering the bacterial cell.

b. Capsule Formation: Some bacteria, like Klebsiella pneumoniae, form a thick polysaccharide

capsule around their cell wall, which hinders antibiotic penetration and enhances resistance.

c. Bypassing the Antibiotic’s Target Pathway: Some bacteria can bypass the metabolic

pathways that antibiotics target, either by using alternative pathways or by producing excess

amounts of the target enzyme.

d. Folate Synthesis Pathway: Sulfonamides and trimethoprim inhibit enzymes in the folate

synthesis pathway. Some resistant bacteria acquire alternative forms of these enzymes or

increase production, allowing the bacteria to continue synthesizing folate despite the

presence of antibiotics.

5. Biofilm Formation: Bacteria in biofilms are inherently more resistant to antibiotics than

planktonic (free-living) bacteria. A biofilm is a complex aggregation of bacteria encased in a

self-produced extracellular matrix.

a. Protective Barrier: The biofilm matrix can prevent antibiotics from penetrating to the

bacterial cells, resulting in reduced effectiveness.

b. Altered Microenvironment: The slow growth and reduced metabolic activity of bacteria in

biofilms can make antibiotics, particularly those targeting actively dividing cells, less

effective.

6. Horizontal Gene Transfer (HGT): Bacteria can acquire antibiotic resistance genes from

other bacteria through horizontal gene transfer. This is a significant way that resistance

spreads rapidly among bacterial populations.

a. Conjugation: The transfer of plasmids (small, circular pieces of DNA) containing

resistance genes between bacteria through direct contact.

b. Transformation: The uptake of free DNA containing resistance genes from the

environment.

c. Transduction: Bacteriophages (viruses that infect bacteria) can transfer resistance genes

from one bacterium to another.

1.4.4.2 Overview of Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to amplify

specific segments of DNA. It allows for the generation of millions to billions of copies of a

particular DNA sequence from a small initial sample, making it easier to study, analyze, and

manipulate. PCR is widely used in various fields such as genetics, microbiology, forensics,

medical diagnostics, and biotechnology due to its precision, sensitivity, and versatility (Shahzad

et al., 2020).

PCR was developed by Kary Mullis in 1983, revolutionizing the field of molecular biology.

Mullis won the Nobel Prize in Chemistry in 1993 for this breakthrough, as PCR made it possible

to replicate small amounts of DNA in a short period without the need for a living organism (like

bacteria) for cloning the DNA (Tayyeb and Basit, 2023).

 Principle of PCR

PCR mimics the natural process of DNA replication, where a double-stranded DNA molecule is

duplicated. However, unlike cellular replication, PCR occurs in vitro (outside of living cells) and

requires specific components and conditions. The core principle of PCR involves the repeated

cycling of heating and cooling to facilitate the denaturation, annealing, and extension of DNA

strands (Shahzad et al., 2020).

Procedure for PCR

PCR involves three main steps, each repeated in cycles (typically 25-40 cycles) to exponentially

amplify the DNA target sequence:

i. Denaturation (94-98°C): The double-stranded DNA is heated to high temperatures to break the

hydrogen bonds between the complementary base pairs, causing the DNA to separate into two

single strands (Aycan and Yildiz, 2024).

ii. Annealing (50-65°C): The reaction mixture is cooled to a lower temperature, allowing the

primers to bind (anneal) to their complementary sequences on the single-stranded DNA

template. The exact annealing temperature depends on the length and sequence of the primers

(Shahzad et al., 2020).

iii. Extension/Elongation (72°C): Taq DNA polymerase synthesizes new DNA strands by adding

dNTPs to the 3’ end of the primers, using the single-stranded DNA as a template. The optimal

temperature for this extension process is around 72°C, the temperature at which Taq

polymerase functions best (Shahzad et al., 2020).

These three steps are repeated multiple times, and with each cycle, the amount of target DNA

doubles, leading to an exponential amplification of the specific DNA region.

Applications of PCR

PCR has a wide range of applications in research, diagnostics, and various industrial fields:

1. Medical Diagnostics: It is used in the detection of infectious diseases like COVID-19, HIV,

tuberculosis, and hepatitis as well as genetic testing for inherited disorders such as cystic

fibrosis or sickle cell anemia (Wang et al., 2024).

2. Forensic Science: PCR is used in DNA profiling for crime scene investigations, paternity

testing, and identification of remains. Also, even small, degraded DNA samples can be

amplified to create a DNA profile (Bukyya et al., 2021).

3. Molecular Biology Research: PCR is essential for cloning DNA fragments, analyzing gene

expression, and studying genetic mutations. It is also used in creating genetically modified

organisms (GMOs) and studying evolutionary relationships between species (Miljuš-Đukić

and Banović Đeri, 2021).

4. Agriculture: PCR is employed to detect genetically modified organisms in crops and foods.

Also, it is used for identifying plant pathogens and selecting desirable genetic traits in

breeding programs (Jamil et al., 2024).

5. Environmental Science: PCR is used to detect microbial contamination in water, soil, and

air. It also helps monitor biodiversity and track the spread of invasive species or endangered

organisms (Banerjee et al., 2021).

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