ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF SOME MULTI-DRUG

RESISTANT GRAM NEGATIVE BACTERIA FROM HOSPITAL EFFLUENT

AND DOMESTIC SEWAGE IN DUTSIN-MA METROPOLIS.

BY

ABDULLAH UMAR HUSSAINI

LSC/2020/13394 DEPARTMENT OF MICROBIOLOGY, FACULTY OF LIFE

SCIENCE,

FEDERAL UNIVERSITY DUTSIN-MA, KATSINA STATE

IN PARTIAL FULFILLMENT AS A REQUIREMENT FOR THE AWARD OF

BACHELOR OF SCIENCE DEGREEE (B.sc) IN MICROBIOLOGY

SUPERVISED

BY

KHALIFAH JAMIL SALEH

0
 DECLARATION

I, ABDULLAH UMAR HUSSAINI with matriculation number LSC/2020/13394 hereby

declare that this research project was carried out by me under the supervision and

guidance of KHALIFAH JAMIL SALEH. It comprises the summary of project reseach

carried out by me. I therefore submit the report work as partial fulfuilment for the

requirements for the award of bachelor of Science in Microbiology, federal university

Dutsin-ma.

_______________________ ____________________________
ABDULLAH UMAR HUSSAINI DATE
(LSC/2020/13394)

1
 CERTIFICATION

This is to certify that this project titled "A ntimicrobial susceptibility patterns of multidrug-

resistant Gram-negative bacteria isolated from hospital effluent and domestic sewage in the

Dutsin-Ma metropolis".was carried out by ABDULLAH UMAR HUSSAINI with

matriculation number LSC/2020/13394 of the department of microbiology has presented

this as a research project write up.

______________________ ______________________
KHALIFAH JAMIL SALEH Date
(Project Supervisor)

_____________________ ______________________
MR. WATA INNOCENT Date
(Project Coordinator)

________________________ ______________________
Mal. UMAR ABDULMALIK Date
(Head of Department)

_____________________ ______________________
Date
(External Examiner)

2
 DEDICATION

This work is dedicated to Almighty ALLAH and my beloved Father Abdullahi Hussaini

Dallaji, my beloved mother, Hajiya Jamila Sa'eed, my supportive supervisor Khalifah

jamil saleh and my beloved sisters and brothers.

3
 ACKNOWNLEDGMENT

All Thanks be to almighty Allah (S.W.A) for his blessings, guidance, protections,

courage and opportunity given to me for the successful completion of my project

research. May his protection and blessings be with us, (amen).May the peace and

blessings of Allah be upon our beloved prophet Muhammad (S.A.W), his family,

companions and those who follow the right path till the Day of Judgment Amen.

I wish to express my thanks to my beloved parents for their moral support toward the

course of this program.

I also wish to express my vote of thanks to the management and Staffs in the department

of Microbiology Federal University Dutsin-ma, Katsina state, especially my project

supervisor Khalifah jamil saleh, my HOD Mal Umar Abdulmalik, Mr. Alabi and Mal.

Adamu Adamu Muhammad for their guidance, help, and support toward the completion

of this report. My deepest gratitude goes to my friends Abubakar tijjani (Daura) and

Hussaini Haruna ( PRO) and friends for their massive support and contribution towards

the success of this project.

4
 ABSTRACT

This study aimed to investigate the antimicrobial susceptibility patterns of multidrug-resistant

Gram-negative bacteria isolated from hospital effluent and domestic sewage in the Dutsin-Ma

metropolis.A total of 40 samples were collected from four locations: General Hospital (GH),

Comprehensive Hospital (CH), Kadangaru (KG), and Darawa (DW). Bacterial isolates were

identified and subjected to antimicrobial susceptibility testing using the Kirby-Bauer disc

diffusion method on Mueller-Hinton agar following CLSI guidelines (2024). This study focused

on three major pathogens: Pseudomonas aeruginosa, Escherichia coli, and Klebsiella

pneumoniae.Thus study identified Pseudomonas aeruginosa as the most prevalent multidrug-

resistant organism, followed by Escherichia coli and Klebsiella pneumoniae. In hospital

effluents, Pseudomonas aeruginosa showed 100% resistance to Augmentin and significant

resistance to Nalidixic acid (80%), Gentamicin (80%), Ciprofloxacin (40%), and Ceporex (60%).

Escherichia coli exhibited high resistance to Augmentin (60%), Nalidixic acid (80%),

Ciprofloxacin (80%), and Ceporex (60%). Klebsiella pneumoniae displayed 100% resistance to

Nalidixic acid and 60% resistance to Ciprofloxacin. Similar resistance patterns were observed in

bacteria isolated from domestic sewage. The high prevalence of multidrug-resistant bacteria in

both hospital effluents and domestic sewage underscores the urgent need for enhanced

wastewater treatment processes, strengthened antibiotic stewardship programs, continuous

surveillance, and effective infection control measures.

5
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

The emergence and spread of multi-drug resistant (MDR) Gram-negative bacteria pose a

significant challenge to public health worldwide. Gram-negative bacteria, such as Escherichia

coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, are notorious for their ability to

develop resistance to multiple antibiotics, rendering conventional treatment ineffective (Paterson

and Bonomo, 2005). This phenomenon has been exacerbated by the selective pressure exerted by

the overuse and misuse of antibiotics in healthcare settings, agriculture, and domestic

environments (Martínez et al., 2007).

Hospital effluents and domestic sewage are recognized as important reservoirs for MDR

bacteria, serving as conduits for the dissemination of these pathogens into the environment

(Baquero et al., 2008). Effluents from healthcare facilities contain high concentrations of

antibiotics, disinfectants, and other pharmaceuticals, providing ideal conditions for the selection

and proliferation of resistant strains (Kümmerer, 2009). Similarly, domestic sewage contributes

to the environmental load of antimicrobial agents through the excretion of antibiotics by humans

and animals (Novo et al., 2013).

Understanding the antimicrobial susceptibility patterns of MDR Gram-negative bacteria isolated

from hospital effluents and domestic sewage is crucial for assessing the extent of environmental

contamination and devising effective control measures. Previous studies have documented the
6
presence of MDR strains in these wastewater sources, highlighting the need for continued

surveillance and intervention (WHO, 2014). However, there remains a paucity of comprehensive

data on the susceptibility profiles of these bacteria in such environments.

Antibiotic resistance is a pressing global health issue that threatens the effectiveness of

antibiotics in treating bacterial infections. Since the discovery of penicillin by Alexander

Fleming in 1928, antibiotics have revolutionized modern medicine, saving millions of lives by

effectively combating bacterial infections. However, the widespread use and misuse of

antibiotics have led to the emergence of antibiotic-resistant bacteria, compromising their efficacy

and posing significant challenges to healthcare systems worldwide (Laxminarayan et al.,

2013).The emergence and spread of antibiotic resistance have far-reaching implications for

healthcare delivery, patient outcomes, and public health. Resistant bacterial infections are

associated with prolonged illness, increased mortality rates, and higher healthcare costs due to

the need for more expensive and intensive treatment regimens (World Health Organization,

2014). Moreover, antibiotic-resistant bacteria can spread within healthcare facilities,

communities, and across international borders, making them a global health threat that requires

coordinated efforts to address (CDC, 2019).In recent years, the rise of multidrug-resistant

bacteria, which are resistant to multiple classes of antibiotics, has further exacerbated the

problem of antibiotic resistance. Multidrug-resistant organisms (MDROs) can cause severe

infections that are difficult to treat, leading to treatment failures and higher rates of morbidity

and mortality (Tacconelli et al., 2018). Additionally, the limited pipeline of new antibiotics and

the slow pace of antibiotic discovery pose challenges in addressing the growing threat of

antibiotic resistance (Theuretzbacher, 2017).

7
Wastewater, including effluents from hospitals and domestic sources, serves as a reservoir for

antibiotic-resistant bacteria and antibiotic resistance genes (Czekalski et al., 2016).These bacteria

and genes can enter the environment through wastewater discharge, potentially impacting human

health and ecosystems (Manaia et al., 2018). Hospital wastewater, in particular, contains high

concentrations of antibiotics and antibiotic-resistant bacteria due to the extensive use of

antibiotics in clinical settings (Yang et al., 2017). Domestic wastewater also contributes to the

environmental burden of antibiotic resistance, with human activities such as agriculture and

improper disposal of pharmaceuticals further exacerbating the problem (Bengtsson-Palme et al.,

2018).

1.2 Statement of Problems

Despite the pivotal role of antibiotics in modern medicine, the emergence and spread of

antibiotic resistance present a significant challenge to public health worldwide. Antibiotic-

resistant bacteria are increasingly prevalent in various environments, including hospital and

domestic wastewater, posing risks to human health and the environment. While previous studies

have highlighted the presence of antibiotic-resistant bacteria in wastewater, there is a need for

comprehensive assessment and understanding of the prevalence, distribution, and characteristics

of these bacteria in different wastewater sources. Furthermore, the effectiveness of wastewater

treatment processes in reducing antibiotic resistance levels and the potential risks associated with

the presence of antibiotic-resistant bacteria in the environment remain areas requiring further

investigation. Addressing these knowledge gaps is crucial for informing strategies to mitigate

antibiotic resistance and safeguard public health.

8
Therefore, the primary problem addressed in this study is to assess antibiotic resistance bacteria

in hospital and domestic wastewater, with implications for public health.

1.3 Justification
The study is justified by its relevance to public health and environmental monitoring, its

alignment with the One Health approach, and its potential to inform antimicrobial stewardship

efforts and policy interventions. Assessing antibiotic-resistant bacteria in wastewater is crucial

for understanding transmission dynamics, identifying environmental risks, and developing

integrated strategies to mitigate antibiotic resistance.

1.4 Aim and objectives of the study

1.4.1 Aim
The aim of this study is to determine the antibiotic susceptibility profile of wastewater from

domestic and hospitals of Dutsin-Ma Metropolis.

1.4.2 Objectives
The specific objectives of the study are to;

1. To isolate and identify bacteria from hospital effluent and domestic sewage of during

2. To determine the antibiotic susceptibility profile of the isolates

3. To determine Multi drug resistant bacteria among the isolates

9
 CHAPTER TWO

2.0. LITERATURE REVIEW

2.1. Introduction
The history of antibiotics spans millennia, with early civilizations like the ancient Egyptians and

Greeks using natural substances such as moldy bread and honey to treat infections. However, the

modern era of antibiotics began with Alexander Fleming's discovery of penicillin in 1928, which

revolutionized medicine by providing a potent weapon against bacterial infections. Following

this breakthrough, the golden age of antibiotic discovery ensued, with researchers isolating and

synthesizing a multitude of antibiotics from various sources, including soil microbes and fungi.

10
This period saw the development of antibiotics such as streptomycin, tetracycline, and

erythromycin, which expanded the arsenal of weapons against infectious diseases. The

widespread use of antibiotics in medicine, agriculture, and animal husbandry led to significant

improvements in public health, with mortality rates from infectious diseases plummeting. .

Despite these challenges, ongoing research and innovation continue to drive the quest for new

antibiotics and strategies to combat antibiotic resistance in the 21st century (Ventola,

2015).Antibiotics are a class of medications used to treat bacterial infections by inhibiting the

growth or killing bacteria (Ventola, 2015). However, overuse and misuse of antibiotics have led

to the development of antibiotic resistance, where bacteria evolve mechanisms to withstand the

effects of antibiotics, rendering them ineffective (Laxminarayan et al., 2013).

The prevalence of multi-drug resistant (MDR) Gram-negative bacteria poses a significant threat

to public health worldwide. The rapid emergence and spread of these pathogens, particularly in

hospital effluents and domestic sewage, complicate treatment regimens and elevate healthcare

costs. Hospital effluents and domestic sewage are key reservoirs for the dissemination of these

resistant strains into the environment, leading to widespread public health implications.

2.2 Antimicrobial Resistance in Gram-Negative Bacteria

Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas

aeruginosa, are known for their intrinsic and acquired resistance mechanisms. Their outer

membrane acts as a barrier to many antibiotics, and they possess efflux pumps and enzymes like

β-lactamases that degrade antibiotics (Paterson and Bonomo, 2005). These bacteria are capable

of acquiring resistance genes through horizontal gene transfer, further compounding the issue

(Martínez et al., 2015).

11
2.2.1 Sources of Multi-Drug Resistant Bacteria
Hospital effluents and domestic sewage are major sources of MDR bacteria. Hospital effluents

contain high concentrations of antibiotics and disinfectants, which apply selective pressure,

promoting the survival and proliferation of resistant strains (Kümmerer, 2009). Domestic

sewage, on the other hand, is a composite of household waste, which includes antibiotics

excreted by humans, thus contributing to the environmental load of antimicrobial agents (Novo

et al., 2013).

2.3 Antimicrobial Susceptibility Patterns

2.3.1 Escherichia coli

Escherichia coli (E. coli) is a common Gram-negative bacterium found in the intestines of

humans and animals. However, pathogenic strains can cause severe infections. MDR E. coli

strains are frequently isolated from hospital effluents and domestic sewage. Studies have shown

a high resistance rate to commonly used antibiotics such as ampicillin, ciprofloxacin, and

cephalosporins (Pitout et al., 2005). Extended-spectrum β-lactamases (ESBL)-producing E. coli

are particularly concerning due to their resistance to third-generation cephalosporins (Bradford,

2001).

Pathogenic Strains: Pathogenic strains of E. coli are classified into different pathotypes based

on their virulence factors and clinical manifestations. Some of the most well-known pathotypes

include enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E.

coli (EHEC), and uropathogenic E. coli (UPEC).

Virulence Factors: Pathogenic strains of E. coli possess various virulence factors that enable

them to colonize host tissues, evade host defenses, and cause disease. These virulence factors
12
include adhesins, toxins, invasins, and secretion systems, which facilitate adherence to host cells,

destruction of cell membranes, and manipulation of host cell processes.

Disease Manifestations: E. coli infections can result in a wide range of clinical manifestations,

depending on the strain and site of infection. Gastrointestinal infections caused by E. coli

typically present with symptoms such as diarrhea, abdominal pain, and fever, while urinary tract

infections may cause symptoms such as dysuria, frequency, and hematuria.

Antibiotic Resistance: Antibiotic resistance is a growing concern in E. coli, particularly among

strains associated with healthcare-associated infections. Resistance to commonly used antibiotics

such as fluoroquinolones, cephalosporins, and trimethoprim-sulfamethoxazole has been

documented, complicating the treatment of E. coli infections.

Public Health Implications: E. coli is a significant public health concern due to its role in

foodborne outbreaks, healthcare-associated infections, and antimicrobial resistance. Proper

hygiene, sanitation, and food safety practices are essential for preventing E. coli infections and

minimizing their impact on public health.

2 3.2 Klebsiella pneumoniae

Klebsiella pneumoniae is another Gram-negative bacterium associated with various infections,

including pneumonia, urinary tract infections, and septicemia. MDR strains of K. pneumoniae

are characterized by their resistance to carbapenems, often mediated by carbapenemase enzymes

such as KPC, NDM, and OXA-48 (Nordmann et al., 2011). Studies have indicated a high

prevalence of carbapenem-resistant K. pneumoniae (CRKP) in hospital settings and effluents,

posing significant treatment challenges (Pitout et al., 2015).

13
Antibiotic Resistance: K. pneumoniae has become increasingly resistant to multiple antibiotics,

posing a significant challenge to healthcare systems worldwide. Resistance mechanisms include

the production of extended-spectrum beta-lactamases (ESBLs), carbapenemases (such as KPC,

NDM, and OXA), and other enzymes that hydrolyze beta-lactam antibiotics. Additionally,

K. pneumoniae can acquire resistance through mutations in target genes, efflux pumps, and

alterations in outer membrane permeability.

Clinical Implications: Multidrug-resistant K. pneumoniae infections are associated with

increased morbidity, mortality, and healthcare costs. These infections are often difficult to treat,

requiring the use of last-line antibiotics such as carbapenems, which themselves are becoming

less effective due to the emergence of carbapenem-resistant strains. Control measures, including

antibiotic stewardship programs, infection prevention strategies, and surveillance of

antimicrobial resistance, are crucial for addressing the threat posed by multidrug-resistant K.

pneumoniae.

2. 3.3 Pseudomonas aeruginosa

Pseudomonas aeruginosa is a notorious opportunistic pathogen, particularly in immune

compromised individuals. Its intrinsic resistance to many antibiotics is due to its low outer

membrane permeability and efflux pumps. MDR P. aeruginosa strains have shown high

resistance to β-lactams, aminoglycosides, and fluoroquinolones (Lister et al., 2009). The

presence of these strains in hospital effluents is concerning as they can easily spread to the

community and environment.

Antibiotic Resistance: P. aeruginosa exhibits intrinsic resistance to many antibiotics due to its

impermeable outer membrane, efflux pumps, and production of enzymes that degrade antibiotics.
14
Additionally, it can acquire resistance through mutations in chromosomal genes or by acquiring

resistance genes through horizontal gene transfer. Multidrug-resistant strains of P. aeruginosa,

especially those resistant to carbapenems, pose significant challenges for treatment.

Clinical Implications: Infections caused by multidrug-resistant P. aeruginosa are associated

with increased morbidity, mortality, and healthcare costs. These infections are difficult to treat,

requiring combination therapy with multiple antibiotics or the use of last-line agents such as

polymyxins. Prevention of P. aeruginosa infections through infection control measures,

antibiotic stewardship, and surveillance of antimicrobial resistance is crucial for mitigating its

impact on public health.

2.4 Wastewater
Wastewater refers to any water that has been used in various human activities and has become

contaminated as a result. It includes water from domestic, industrial, commercial, and

agricultural sources. Wastewater can contain a wide range of contaminants, including organic

matter, pathogens, nutrients, heavy metals, and synthetic chemicals. Improper management of

wastewater can lead to pollution of water bodies, soil, and air, posing significant risks to human

health and the environment.

Wastewater, which includes domestic, industrial, and hospital effluents, serves as a reservoir for

diverse microbial communities, including ARB and ARGs (Baquero et al., 2008). The discharge

of untreated or inadequately treated wastewater into the environment can introduce antibiotic-

resistant bacteria and genes into natural ecosystems, where they may persist and disseminate

through water bodies, soil, and food chains (Gaze et al., 2013). Furthermore, wastewater

treatment plants may serve as hotspots for the selection and proliferation of antibiotic-resistant
15
bacteria due to the presence of antibiotics and other selective agents in wastewater (Michael et

al., 2013).

Wastewater is a ubiquitous byproduct of human activities, encompassing a diverse array of liquid

and solid waste materials discharged from various sources. It includes water contaminated

through domestic, industrial, commercial, agricultural, and hospital activities, reflecting the

broad spectrum of human interactions with the environment (García et al., 2016).

2.4.1 Composition of wastewater

The composition of wastewater can vary depending on its source and the activities it has been

exposed to. However, typical components of wastewater include;

Organic Matter: This includes biodegradable substances such as human and animal waste, food

scraps, oils, and grease. Organic matter serves as a food source for microorganisms and can

contribute to oxygen depletion in water bodies if not properly treated.

Nutrients: Wastewater contains nutrients such as nitrogen and phosphorus, which originate from

human waste, detergents, fertilizers, and organic matter. Excessive nutrient levels in water bodies

can lead to eutrophication, causing algal blooms, oxygen depletion, and ecosystem degradation.

Pathogens: Wastewater may contain disease-causing microorganisms such as bacteria, viruses,

and parasites, originating from human and animal waste. These pathogens can pose health risks

to humans and animals if wastewater is not adequately treated before discharge or reuse.

Suspended Solids: These are solid particles suspended in wastewater, including soil, sediment,

debris, and organic matter. Suspended solids can cause turbidity in water bodies, interfere with

aquatic ecosystems, and contribute to the clogging of wastewater treatment systems.

16
Chemical Contaminants: Wastewater can contain various chemical pollutants, including heavy

metals, pesticides, pharmaceuticals, industrial chemicals, and household products. These

contaminants may originate from industrial discharges, agricultural runoff, and domestic

activities, posing risks to human health and the environment.

PH and Temperature: The pH of wastewater can range from acidic to alkaline depending on its

composition and source. Temperature can also vary depending on the wastewater source and

environmental conditions. Fluctuations in pH and temperature can affect the survival and growth

of aquatic organisms.

Dissolved Oxygen: Wastewater may contain dissolved oxygen, which is essential for supporting

aquatic life. However, organic matter decomposition and microbial activity can deplete oxygen

levels in water bodies, leading to hypoxic or anoxic conditions that harm aquatic organisms.

2.4.2 Treatment of wastewater

The treatment of wastewater involves several processes designed to remove contaminants and

improve water quality before its discharge into the environment or its reuse for various purposes.

The primary objectives of wastewater treatment are to protect public health, safeguard the

environment, and promote sustainable water management. The treatment process typically

consists of the following stages:

Preliminary Treatment: In this stage, large debris, grit, and other coarse solids are removed

from the wastewater through screening and sedimentation processes. This helps protect

downstream treatment units from damage and prevents clogging of pipes and equipment.

17
Primary Treatment: During primary treatment, suspended solids and organic matter are further

removed from the wastewater through sedimentation and flotation processes. Gravity settling

allows heavier particles to settle at the bottom of tanks, forming sludge, while lighter particles

are skimmed off the surface. This step reduces the biochemical oxygen demand (BOD) and total

suspended solids (TSS) in the wastewater.

Secondary Treatment: Secondary treatment aims to remove dissolved and colloidal organic

matter, as well as nutrients such as nitrogen and phosphorus, from the wastewater. This is

typically achieved through biological processes, such as activated sludge treatment, trickling

filters, or biological nutrient removal (BNR) systems. Microorganisms, including bacteria and

protozoa, metabolize organic pollutants and convert them into carbon dioxide, water, and

microbial biomass.

Tertiary Treatment: Tertiary treatment involves additional processes to further improve the

quality of treated wastewater before its discharge or reuse. This may include advanced filtration

techniques such as sand filtration, membrane filtration, or adsorption onto activated carbon.

Tertiary treatment can remove residual contaminants, pathogens, and nutrients to meet specific

water quality standards or to enable safe reuse of the treated wastewater for irrigation, industrial

processes, or environmental restoration.

Disinfection: Disinfection is a critical step in wastewater treatment to inactivate or destroy

remaining pathogens and microorganisms in the treated effluent. Common disinfection methods

include chlorination, ultraviolet (UV) irradiation, ozonation, and chloramination. Disinfected

wastewater can be safely discharged into surface waters or reused for non-potable purposes, such

as irrigation or industrial cooling.

18
Sludge Treatment and Disposal: Sludge generated during wastewater treatment processes,

including primary and secondary sedimentation and sludge dewatering, requires further

treatment before disposal or beneficial reuse. Sludge treatment options may include anaerobic

digestion, aerobic composting, dewatering, and thermal drying. The treated sludge can be used as

soil conditioner, fertilizer, or disposed of in a landfill or through other appropriate methods.

2.4.3 Regulations and management of wastewater

Regulation and management of wastewater involve the development and implementation of

policies, standards, and practices to ensure the safe and effective treatment, disposal, and reuse of

wastewater while protecting human health and the environment. Key aspects of wastewater

regulation and management include:

Legislative Framework: Governments at the national, regional, and local levels enact laws,

regulations, and ordinances to govern the management of wastewater. These legal frameworks

establish requirements for wastewater discharge, treatment, quality standards, monitoring,

enforcement, and compliance.

Permitting and Licensing: Regulatory agencies issue permits and licenses to wastewater

treatment facilities, industries, municipalities, and other entities involved in wastewater

management. These permits specify the conditions, limits, and requirements for wastewater

discharge, effluent quality, pollutant levels, and operational practices.

Water Quality Standards: Regulatory authorities establish water quality standards that define

the acceptable levels of pollutants, contaminants, and pathogens in treated wastewater effluent

and receiving water bodies. These standards aim to protect human health, aquatic ecosystems,

19
and beneficial uses of water resources, such as drinking water supply, recreation, and aquatic

habitat.

Public Participation and Stakeholder Engagement: Wastewater regulation and management

processes often involve public participation, stakeholder engagement, and consultation with

affected communities, environmental organizations, industry representatives, and other

stakeholders. Public input helps inform decision-making, policy development, and planning for

wastewater infrastructure projects.

Technology and Innovation: Regulatory agencies promote the adoption of advanced

technologies, best practices, and innovative solutions for wastewater treatment, reuse, and

pollution prevention. This may include incentives, grants, funding programs, and technical

assistance to support research, development, and implementation of sustainable wastewater

management practices.

International Cooperation: Wastewater regulation and management efforts often extend

beyond national boundaries, requiring collaboration and cooperation among countries,

international organizations, and stakeholders. International agreements, treaties, and initiatives

address transboundary water pollution, marine pollution, and global water quality challenges,

fostering cooperation in addressing shared wastewater management issues.

2.5 Impact of Hospital effluent and Domestic sewage.

The discharge of hospital effluents and domestic sewage containing MDR bacteria into the

environment has several implications. Firstly, it leads to the contamination of water bodies,

which are sources of drinking water and irrigation (Baquero et al., 2008). Secondly, it facilitates

the horizontal transfer of resistance genes among environmental bacteria, creating a reservoir of
20
resistance (Martínez, 2009). Lastly, it poses risks to public health as these bacteria can infect

humans and animals directly through water and food or indirectly via environmental contact.

2.5.1 Strategies to Mitigate the Spread of MDR Bacteria.

Advanced treatment methods for hospital effluents, such as membrane bioreactors, advanced

oxidation processes, and constructed wetlands, can significantly reduce the load of antibiotics

and MDR bacteria before discharge (Verlicchi et al., 2010). Implementing such technologies is

crucial for controlling the spread of resistance.

Monitoring and Surveillance: Continuous monitoring and surveillance of MDR bacteria in

hospital effluents and domestic sewage are essential. This includes regular sampling and

antimicrobial susceptibility testing to track resistance patterns and implement appropriate control

measures (WHO, 2014).

Antimicrobial Stewardship: Antimicrobial stewardship programs in healthcare settings aim to

optimize the use of antibiotics to reduce the selective pressure that drives the emergence of

resistance (Dyar et al., 2017). Such programs should be coupled with public education on the

prude

2.6 Antibiotic resistant

Antibiotic resistant is the ability of microorganisms to withstand the effects of antibiotics, has

become a critical global health issue. The widespread use and misuse of antibiotics in human

medicine, agriculture, and animal husbandry have accelerated the development and spread of

antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) (Ventola, 2015). As a

result, many once-effective antibiotics are now ineffective in treating bacterial infections, leading

to increased morbidity, mortality, and healthcare costs (CDC, 2019).

21
2.6.1 Mechanism of antibiotic resistant
Enzymatic Inactivation: Enzymatic inactivation refers to a mechanism of antibiotic resistance

employed by bacteria, where they produce enzymes that modify or degrade antibiotics, rendering

them ineffective. One example of enzymatic inactivation is the production of β-lactamases,

which hydrolyze the β-lactam ring of β-lactam antibiotics such as penicillins and cephalosporins,

thereby inactivating these antibiotics and conferring resistance to bacterial strains. This

mechanism is widespread among bacteria and contributes significantly to the clinical challenge

of treating infections caused by antibiotic-resistant pathogens.

Alteration of Target Site: Alteration of target site is a mechanism of antibiotic resistance where

bacteria modify the target sites of antibiotics, preventing the drugs from binding effectively and

exerting their antimicrobial activity. This alteration can occur through mutations in bacterial

genes encoding antibiotic targets or by acquiring resistant versions of these genes through

horizontal gene transfer. By changing the structure or function of the target site, bacteria can

evade the action of antibiotics, leading to treatment failure and the persistence of infections. This

mechanism highlights the adaptive capabilities of bacteria and underscores the need for

continued surveillance and development of novel antimicrobial agents to combat resistance.

Reduced Permeability or Increased Efflux: Reduced permeability or increased efflux is a

mechanism of antibiotic resistance where bacteria develop strategies to prevent antibiotics from

entering the cell or pump them out of the cell, thereby reducing their intracellular concentration

and efficacy. This mechanism can involve changes in the bacterial cell membrane structure to

limit antibiotic penetration or the upregulation of efflux pump proteins to actively remove

antibiotics from the cell. By limiting antibiotic accumulation inside the bacterial cell, this

22
mechanism helps bacteria survive exposure to antibiotics and contributes to the development of

resistance. Understanding and targeting these resistance mechanisms are crucial for the

development of effective antimicrobial strategies.

Target Site Modification: Target site modification is a mechanism of antibiotic resistance

where bacteria alter the structure of their target sites, such as ribosomes or enzymes, to reduce

the binding affinity of antibiotics. This modification prevents antibiotics from effectively

inhibiting their target molecules, thereby diminishing their antimicrobial activity. Target site

modification can occur through various mechanisms, including mutations in genes encoding

antibiotic targets or the acquisition of resistant versions of these genes through horizontal gene

transfer. By evading the action of antibiotics, bacteria can survive and proliferate in the presence

of these drugs, leading to the development of antibiotic resistance.

Biofilm Formation: Biofilm formation is a complex process by which bacteria adhere to

surfaces and produce an extracellular matrix composed of polysaccharides, proteins, and DNA,

forming a structured community known as a biofilm. Within biofilms, bacteria exhibit increased

resistance to antibiotics compared to their planktonic counterparts, making biofilm-associated

infections notoriously difficult to treat. The protective matrix of biofilms acts as a physical

barrier that limits antibiotic penetration and shields bacteria from host immune responses.

Moreover, the metabolic and physiological changes induced by biofilm growth can render

bacteria less susceptible to antimicrobial agents. Biofilm-associated antibiotic resistance poses a

significant challenge in clinical settings, contributing to the persistence of chronic infections and

the recurrence of treated conditions.

23
 Quorum Sensing: Quorum sensing is a communication mechanism used by bacteria to

coordinate gene expression in response to changes in population density. Through the production

and detection of signaling molecules called autoinducers, bacteria can regulate the expression of

genes involved in various cellular processes, including virulence, biofilm formation, and

antibiotic resistance. Quorum sensing enables bacteria to synchronize their behaviors and

collectively respond to environmental cues, enhancing their adaptability and survival in diverse

ecological niches. In the context of antibiotic resistance, quorum sensing can modulate the

expression of genes encoding antibiotic efflux pumps, detoxification enzymes, and biofilm-

associated factors, allowing bacteria to mount coordinated responses to antibiotic exposure.

Disrupting quorum sensing pathways represents a potential strategy for attenuating bacterial

virulence and enhancing the efficacy of antibiotic therapy.

Antibiotic Modification or Sequestration: Antibiotic modification or sequestration refers to

bacterial mechanisms that alter or sequester antibiotics to reduce their efficacy. Bacteria may

produce enzymes that chemically modify antibiotics, rendering them inactive or less effective

against bacterial targets. Alternatively, bacteria may sequester antibiotics within cellular

compartments, such as vesicles or vacuoles, thereby reducing their availability to exert

antimicrobial activity. These mechanisms contribute to bacterial survival in the presence of

antibiotics and can confer resistance to multiple classes of antimicrobial agents. By modifying or

sequestering antibiotics, bacteria can evade the detrimental effects of antibiotic exposure and

persist in hostile environments, posing challenges for antimicrobial therapy and infection control

efforts.

24
2.6.2 Emergence of antibiotic resistant
The emergence of antibiotic resistant refers to the process by which bacteria develop

mechanisms to withstand the effects of antibiotics, rendering them less susceptible or resistant to

treatment it can be traced back to the introduction of antibiotics into clinical practice in the mid-

20th century. Over time, the overuse and inappropriate use of antibiotics have exerted selective

pressure on bacterial populations, favoring the survival and proliferation of resistant strains

(Martinez, 2008).The emergence of antibiotic resistance phenomenon has occurred due to the

selective pressure exerted by the widespread use and misuse of antibiotics in various settings,

including human healthcare, agriculture, and animal husbandry. Over time, bacteria have

evolved various mechanisms, such as enzymatic inactivation, target site alterations, reduced

permeability, and efflux pump systems, to counteract the effects of antibiotics. Additionally,

horizontal gene transfer mechanisms, such as conjugation, transformation, and transduction, have

facilitated the spread of antibiotic resistance genes among bacterial populations, contributing to

the global dissemination of resistant strains. The emergence of antibiotic resistance poses

significant challenges to public health, as it compromises the effectiveness of antibiotic therapy,

leading to increased morbidity, mortality, and healthcare costs. Addressing the emergence of

antibiotic resistance requires a multifaceted approach, including prudent antibiotic use, infection

prevention and control measures, surveillance of resistant pathogens, and the development of

new antimicrobial agents and treatment strategies.

2.6.3 Significance of antibiotic in public health

The significance of antibiotic resistance in public health cannot be overstated. Antibiotics are

essential for treating bacterial infections and have contributed significantly to reducing morbidity

and mortality from infectious diseases. However, the emergence and spread of antibiotic-
25
resistant bacteria undermine the effectiveness of these life-saving drugs, posing serious threats to

public health. Antibiotic-resistant infections are associated with longer hospital stays, increased

treatment failure rates, and higher mortality rates compared to infections caused by susceptible

bacteria.

CHAPTER THREE

3.0 METHODOLOGY

3.1 Study Area

The study was carried out in Dutsin-Ma Local Government Area, Katsina State and samples

were collected within Dutsin-Ma Metropolis. It is located on Latitude 12° 27’18” N and

26
longitude 7° 29’29” E and has its headquarters in the town of Dutsin-Ma. It has an estimated

area of 527km 2 (203sqkm) and a population of 169,671 as at 2006 census. The Local

Government is bounded by Kurfi and Charanchi Local Governments to the North, Kankia Local

Government to the East, Safana and Dan- Musa Local Governments to the West, and Matazu

Local Government to the South. The people are predominantly farmers, cattle rearers and traders.

27
3.2 Sample Collection:
A total of 40 wastewater samples were systematically collected from hospital effluent and

domestic sewage sources in Dutsin-Ma Metropolis using sample container.

3.3 Sample analysis

3.3.1 Isolation
Isolation of the bacterial strains was carried out using selective medias such as; Macconkey agar

for the isolation of Klebsiella pneumoniae, Centrimide agar for the isolation of Pseudomonas

aeruginnosa and Eosine Methylene Blue Agar for the isolation of Escherichia coli. Each sample

was cultured for bacteria by direct streaking of 0.5ml of it on different media and incubated for

24 hours at 37°C. After the incubation period positive colonies were detected which appear to

have different morphology and then the colonies were sub-cultured on the same media in another

petri dishes and still incubated for 24 hours at 37°C in order to get pure culture of the isolates.

3.4 Identification

4.4.1 Gram’s Staining.

These tests was done according to (Nester et al; 2007). The Gram stain is by far the most widely

used procedure for staining bacteria and separating it into two major groups: Gram (+) positive

and Gram (-) negative. The isolates were smear thin film over a clean glass slide and allow to air

dry, then the glass slides were heat fix by passing it over a Bunsen flame thrice, the smear film

was flooded with crystal violet and leave for 60 seconds. To the slides been wash off with water

and flood the stain again with lugol’s iodine and leave for 60 seconds. The slides been wash off

again with water and then were decolorized with acetone (decolourizer) for a second, then wash

28
off again with water and they were flodded with safranin (counter stain) for 60 seconds and they

been wash off with water and allow to air dry.

4.4.2 Microscopic:
The slides were view under a microscope which they show a clear distinction between the

purple-colored Gram-positive bacteria and the red-colored Gram-negative bacteria. This help to

identify the different types of bacteria present in the sample.

4.5 Biochemical test

The isolated bacteria were identified through the following biochemical tests (IMVIC).

4.5.1 Indole test:

The indole test was conducted to determine the ability of the bacteria to produce indole from the

amino acid tryptophan were a sterile wire loop was used to transfer a small amount of the

bacteria colony into a test tube containing 5ml sterile peptone water and incubated for about

24hours.After the incubation period few drops of kovac‟s reagent was added into the 24hours

peptone water culture and shake gently, the appearance of red ring above the peptone indicates

positive indole production while if there is no color change indicates negative indole production.

4.5.2 Methyl red test

The methyl red test was used to identify bacteria capable of performing mixed acid fermentation

were a sterile wire loop was used to transfer a small amount of the bacteria colony into a test

tube containing 5ml sterile MR-VP broth and incubated for about 24hours. After the incubation

period few drops of methyl red indicator were drop into the test tube and shake gently, The

appearance of dark ring or red color above indicates the production of stable acid through mixed

acid fermentation.
29
4.5.3 Voges-Proskauer test
The voges-proskauer test was used to detect the presence of acetoin, a product of glucose

fermentation, were a sterile wire loop was used to transfer a small amount of the bacterial colony

into a test tube containing 5ml of MR-VP broth and incubated for 24hours, after the incubation

period few drops of alpha-naphthol and few drops of potassium hydroxide were added into the

test tube and shake gently, The development of dark or red ring above indicate a positive result

confirming the presence of acetoin, lack of color change indicate a negative result.

4.5.4 Citrate test

The citrate test was performed to determine the ability of the bacteria to used citrate as their sole

carbon source, were the bacterial sample was streaked onto a simmons citrate agar slant using

sterile wire loop and incubated for 24hours, After the incubation period the appearance of change

in color of the medium from green to blue indicates positive result showing that the bacteria

could utilize citrate and produce alkaline by products.

4.5.5 Urease test

The urease test was performed to determine the ability of the bacteria to produce urease, were

prepared urea broth was added into a test tube and inoculated with the bacteria the test tube were

incubated for 24hours, positive result as change of color to pink which indicates that the bacteria

can produce urease if not the broth still remain clear.

30
4.5.6 Oxidase test
The oxidase test was perform to determine if the bacteria produce enzyme cytochrome oxidase, a

small loop of the bacteria was taken from the agar plate using sterile wire loop and smear into an

oxidase strip containing 1% Tetramethyl paraphenylene diamine dihydrochloride, and wait for

30 seconds strip color change to dark purple indicates positive result.

4.6 Antibiotic susceptibility test

The disc diffusion method (Kirby-Bauer method) was performed to evaluate the antimicrobial

susceptibility of the bacterial isolates, the bacterial suspension was prepared by adjusting the

turbidity to match the 0.5 Mcfarland standard, A sterile swab was dipped into the bacterial

suspension and the excess liquid was removed by pressing the swab against the inside of the test

tube. The surface of Oxoid Mueller-Hinton agar (Difco Laboratories, Detroit, Mich USA) plates

was then uniformly inoculated by streaking the swab all over the plate.

Commercially prepared antibiotics impregnated discs, containing the following antibiotics:

Augmentin (30µg), Nalidixic acid (30µg), Septrin (30µg), Gentamycin (10µg), Ciprofloxacin

(10µg), Ceporex (10µg) were aseptically placed on the inoculated plates and incubated at 37oC

for 24hrs. The diameter of the zone of clearance (including the diameter of the disk) was

measured to the nearest whole millimeter and interpreted on the basis of CLSI guideline (CLSI,

2024).

31
4.7 Identification of multi-drug resistant bacteria (MDR).
The identification of multi-drug resistant bacteria was carried out from the antibiotic

susceptibility test result, were any bacteria that show resistant to three or more different classes

of antibiotics were classified as multi-drug resistant bacteria (MDR)

CHAPTER FOUR

4.0 RESULT

4.1 Distribution of samples base on the different locations.

Table.1 shows the distribution of the 40 samples collected from four locations: General Hospital

(GH), Comprehensive Hospital (CH), Kadangaru (KG), and Darawa (DW). Each location

contributed an equal number of samples (10), representing 25%of the total samples. This equal

distribution ensures a balanced representation of different environmental sources, allowing for a

comprehensive analysis of antimicrobial resistance patterns in the study area.

4.2 The microscopic features, gram reaction colonial morphology of the bacteria isolated
From hospital effluents.

Table.2 provides details on the microscopic features, Gram reaction, and colonial morphology of

bacteria isolated from hospital effluents. The isolates were all Gram-negative, rod-shaped

32
(bacilli), and exhibited distinct colonial characteristics, such as metallic green sheen indicating

lactose fermentation (commonly associated with E. coli) and greenish colonies with a grape-like

odor due to pyocyanin production (indicative of Pseudomonas aeruginosa). These characteristics

are crucial for the preliminary identification of bacterial isolates before further biochemical

testing.

4.3 The microscopic features, gram reaction colonial morphology of the bacteria isolated
From Domestic sewages.

Table.3 presents the microscopic features, Gram reaction, and colonial morphology of bacteria

isolated from domestic sewages. The isolates showed consistent traits with those from hospital

effluents, such as being Gram-negative bacilli and exhibiting colonial features like pinkish

mucoid colonies due to lactose fermentation and greenish colonies due to pyocyanin production.

This similarity suggests that domestic sewage also harbors significant levels of multidrug-

resistant Gram-negative bacteria.

4.4 Biochemical tests of Pseudomonas aeruginnosa (IMVIC).

Table.4 Outline the results of IMVIC biochemical tests for Pseudomonas aeruginosa isolates.

All tested isolates were negative for indole, methyl red, and Voges-Proskauer tests, but positive

for citrate utilization. These results are consistent with the known biochemical profile of

Pseudomonas aeruginosa, supporting their identification and confirming their presence in both

hospital effluents and domestic sewage.

4.5 Biochemical tests of Escherichia coli (IMVIC).

Table.5 presents the IMVIC test results for Escherichia coli isolates. All isolates were positive

for indole and methyl red tests, and negative for Voges-Proskauer and citrate utilization tests.
33
This biochemical profile aligns with the expected characteristics of E. coli, validating the

identification of these isolates in the collected samples.

4.6 Biochemical test of Klebsiella pneumonia (IMVIC).

Table.6 shows the IMVIC test results for Klebsiella pneumoniae isolates. All isolates were

negative for indole and methyl red tests but positive for Voges-Proskauer and citrate utilization

tests. These results are characteristic of Klebsiella pneumoniae, confirming their identification in

both hospital effluents and domestic sewage samples.

4.7 Distribution of the positive isolates based on the samples collected.

Table details the number of positive bacterial isolates from the total samples collected at each

location. The General Hospital (GH) had the highest percentage of positive isolates (70%),

followed by Comprehensive Hospital (CH) with 50%, Kadangaru (KG) with 40%, and Darawa

(DW) with 30%. These findings suggest that hospital effluents, particularly from larger facilities

like the General Hospital, may be significant reservoirs of multidrug-resistant bacteria.

4.8 Number of percentage (%) susceptibility and resistant of P. aeruginosa, E. coli and
K. pneumoniae isolated from hospital effluents.

Table.8 Provides the susceptibility and resistance patterns of Pseudomonas aeruginosa,

Escherichia coli, and Klebsiella pneumoniae isolated from hospital effluents against various

antibiotics. The results indicate high resistance rates among all three bacteria, particularly to

Augmentin, Nalidixic acid, and Ciprofloxacin. These findings highlight the challenge of treating

infections caused by these resistant bacteria in hospital settings.

4.9 Number of percentage (%) susceptibility and resistant of P. aeruginosa, E. coli and
K. pneumoniae isolated from Domestic sewages.
34
Table.10 shows the susceptibility and resistance patterns of the same bacteria isolated from

domestic sewage. The resistance rates were also high, particularly for Pseudomonas aeruginosa

and Klebsiella pneumoniae against Augmentin, Nalidixic acid, and Ciprofloxacin. The presence

of such resistant bacteria in domestic sewage indicates the potential risk of environmental spread

of antimicrobial resistance.

4.10 Multi-drug resistant pattern of P. aeruginosa, E. coli and K. pneumoniae isolated

From Hospital effluents.

Table.9 outlines the multi-drug resistant patterns observed in the bacterial isolates from hospital

effluents. Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae exhibited

resistance to multiple antibiotics, with some isolates showing resistance to up to five different

antibiotics. This multi-drug resistance complicates treatment options and underscores the need

for vigilant monitoring and management of antibiotic use in healthcare settings.

4.11 Multi-drug resistant pattern of P. aeruginosa, E. coli and K. pneumoniae isolated

From Domestic sewages.

Table.11 details the multi-drug resistant patterns of bacteria isolated from domestic sewage.

Pseudomonas aeruginosa and Klebsiella pneumoniae showed resistance to various combinations

of antibiotics, similar to the patterns observed in hospital effluents. The presence of multi-drug

resistant bacteria in domestic sewage suggests a potential pathway for the dissemination of

resistance into the broader environment, necessitating improved wastewater management

practices.

35
Table 1. Distribution of samples base on the different locations.

SAMPLE LOCATION NO. OF SAMPLES PERCENTAGE (%)

GH 10 25

CH 10 25

KG 10 25

DW 10 25

TOTAL 40 100

KEYS; GH = General hospital

CH = Comprehensive hospital
KG = Kadangaru
DW = Darawa

36
Table 2. The microscopic features, gram reaction colonial morphology of the bacteria
isolated from hospital effluents.

Samples Microscopic G/reaction Colonial morphology Organism

Features idenitfied

GH1 Rod shaped (bacilli) -Ve Metallic green sheen, indicates lactose E.coli
fermentation.

GH2 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor.

GH3 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor.

GH4 Rod shaped (bacilli) -Ve Pinkish colonies with mucoid, due to K. pneumoniae
lactose fermentation.

GH5 Rod shaped (bacilli) -Ve Metallic green sheen, indicates lactose E. coli
fermentation.

GH6 Rod shaped (bacilli) -Ve Metallic green sheen, indicates lactose E. coli
fermentation.

GH7 Rod shaped (bacilli) -Ve Metallic green sheen, indicates lactose E. coli
fermentation.

CH1 Rod shaped (bacilli) -Ve Metallic green sheen, indicates lactose E. coli
fermentation.

CH2 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor.

CH3 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor.

CH4 Rod shaped (bacilli) -Ve Pinkish colonies with mucoid, due to K. pneumoniae
lactose fermentation

CH5 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor

37
KEYS; GH = General hospital, CH = Comprehensive hospital, -Ve = Negative

Table 3. The microscopic features, gram reaction colonial morphology of the bacteria

isolated from Domestic sewages.

Samples Microscopic features G/reaction Colonial morphology Organism

identified
DW1 Rod shaped (bacilli) -Ve Pinkish colonies with mucoid, due K. pneumoniae
to lactose fermentation

DW2 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor

DW3 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor

KG1 Rod shaped (bacilli) -Ve Pinkish colonies with mucoid, K. pneumoniae
due to lactose fermentation

KG2 Rod shaped (bacilli) -Ve Pinkish colonies with mucoid, K. pneumoniae
due to lactose fermentation

KG3 Rod shaped (bacilli) -Ve Greenish colonies, due to pyocyanin P. aeruginosa
production with a grape-like odor

KG4 Rod shaped (bacilli) -Ve Metallic green sheen, indicates lactose E. coli
fermentation.

KEYS; KG = Kadangaru
DW =Darawa
-Ve = Negative

38
Table. 4 Biochemical tests of Pseudomonas aeruginosa.

Samples Indole Methyl red Voges Citrate Oxidase

proskauer
GH2 -ve -ve -ve +ve +ve

GH3 -ve -ve -ve +ve +ve

DW2 -ve -ve -ve +ve +ve

DW3 -ve -ve -ve +ve +ve

KG3 -ve -ve -ve +ve +ve

CH2 -ve -ve -ve +ve +ve

CH3 -ve -ve -ve +ve +ve

CH5 -ve -ve -ve +ve +ve

KEYS; GH= General hospital

DW= Darawa
KG = Kadangaru
CH= Comprehensive hospital

39
Table. 5 Biochemical tests of Escherichia coli (IMVIC).

Samples Indole Methyl red Voges Citrate

proskauer
GH1 +ve +ve -ve -ve

GH5 +ve +ve -ve -ve

GH6 +ve +ve -ve -ve

GH7 +ve +ve -ve -ve

KG4 +ve +ve -ve -ve

CH1 +ve +ve -ve -ve

KEYS; GH= General hospital

KG = Kadangaru
CH= Comprehensive hospital

40
Table. 6 Biochemical test of Klebsiella pneumoniae

Samples Indole Urease Voges proskauer Citrate

GH4 -Ve +Ve +Ve +Ve

DW1 -Ve +Ve +Ve +Ve

KG1 -Ve +Ve +Ve +Ve

KG2 -Ve +Ve +Ve +Ve

CH4 -Ve +Ve +Ve +Ve

KEYS; GH = General hospital

CH = Comprehensive hospital
KG = Kadangaru
DW = Darawa
-Ve = Negative

41
Table.7 Distribution of the positive isolates based on the samples collected.

Samples No. of samples Positive isolates Positive case (%)

GH 10 7 70%

CH 10 5 50%

DW 10 3 30%

KG 10 4 40%

TOTAL 40 19

KEYS; GH = General hospital

CH = Comprehensive hospital
KG = Kadangaru
DW = Darawa

42
Table. 8 Number of percentage (%) susceptibility and resistant of P. aeruginosa, E. coli
and K. pneumoniae isolated from hospital effluents.

Antibiotics P. aeruginosa E. coli K. pneumoniae

S R S R S R
AUG 5(100) 0 2(40) 3(60) 0 2(100)

NA 1(20) 4(80) 1(20) 4(80) 0 2(100)

SXT 3(60) 2(40) 4(80) 1(20) 1(50) 1(50)

CN 1(20) 4(80) 4(80) 1(20) 1(50) 1(50)

CPX 3(60) 2(40) 1(20) 4(80) 2(100) 0

CEF 2(40) 3(60) 2(40) 3(60) 1(50) 1(50)

KEYS; AU = Augumentin
NA = Nalidixic acid
SXT = Septrin
CN = Gentamicin
CPX = Ciprofloxacin
CEF = Ceporex

43
Table. 9 Number of percentage (%) susceptibility and resistant of P. aeruginosa, E. coli
and K. pneumonia isolated from Domestic sewages.

Antibiotic P. aeruginosa E. coli K. pneumonia

s S R S R S R
AUG 0 3(100) 0 1(100) 2(66.67) 1(33.33)

NA 2(66.67) 1(33.33) 1(100) 0 0

3(100)

SXT 2(66.67) 1(33.33) 1(100) 0 3(100) 0

CN 2(66.67) 1(33.33) 1(100) 0 3(100) 0

CPX 1(33.33) 2(66.67) 0 1(100) 1(33.33) 2(66.67)

CEF 0 3(100) 0 1(100) 3(100) 0

KEYS; AU = Augumentin
NA = Nalidixic acid
SXT = Septrin
CN = Gentamicin
CPX = Ciprofloxacin
CEF = Ceporex

44
Table.10 Multi-drug resistant pattern of P. aeruginosa, E. coli and K. pneumoniae
isolated from hospital effluents

Organisms Multi antibiotic resistant pattern

P. aeruginosa AU – NA – CEF

E. coli NA – CN – CPX

P. aeruginosa AU – NA – CN

K. pneumonia AU – NA – CN – CEF

P. aeruginosa AU – NA – CN – SXT

K. pneumonia AU – NA – SXT – CPX

E. coli AU – NA – CPX – CEF

P. aeruginosa AU – CN – SXT – CPX – CEF

E. coli AU – NA – SXT – CPX – CEF

P. aeruginosa AU – NA – CN – CPX – CEF

KEYS; AU = Augumentin
NA = Nalidixic acid
SXT = Septrin
CN = Gentamicin
CPX = Ciprofloxacin
CEF = Ceporex

45
Table.11 Multi-drug resistant pattern of P. aeruginosa, E. coli and K. pneumoniae
isolated from domestic sewages.

Organisms Multi antibiotic resistant pattern

P. aeruginosa AU – CPX – CEF

P. aeruginosa AU – SXT – CEF

K. pneumoniae AU – NA – CPX

P. aeruginosa AU – NA – CN – CPX –CEF

KEYS; AU = Augumentin
NA = Nalidixic acid
SXT = Septrin
CN = Gentamicin
CPX = Ciprofloxacin
CEF = Ceporex

46
 CHAPTER FIVE

5.0 DISCUSSION
This study investigate the antimicrobial susceptibility patterns of multidrug resistant gram-

negative bacteria isolated from hospital effluent and domestic sewage dutsin-ma metropolis. The

distribution of samples was evenly spread across four locations: General Hospital (GH),

Comprehensive Hospital (CH), Kadangaru (KG), and Darawa (DW), with each contributing 25%

of the total samples, a total of 40 samples were collected, with varying positive isolate rates: GH

(70%), CH (50%), DW (30%), and KG (40%). This study identified Pseudomonas aeruginosa,

Escherichia coli and Klebsiella pneumonia as the predominant multidrug-resistant gram negative

bacteria. All isolates were gram negative rods with distinct colonial morphologies indicating

lactose fermentation (metallic green sheen, pinkish mucoid colonies and pyocyanin production

which shows greenish colonies with a grape-like odor).

The AST was done by using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar and

interpreted through CLSI guidelines 2024. Pseudomonas aeruginosa isolated from hospital

effluent exhibited 100% resistance to Augmentin and Significant resistance to Nalidixic acid

80%, Gentamicin 80%, Ciprofloxacin 40%, and Ceporex 60%, Escherichia coli high resistance

to Nalidixic acid 80%, Ciprofloxacin 80% and Ceporex 60%, Klebsiella pneumonia displayed

100% Nalidixic acid and 60% resistance to Ciprofloxacin. While those isolated from domestic

sewage Pseudomonas aeruginosa shows 100% resistance to Augmentin and Ceporex with

47
significant resistance to Ciprofloxacin 66.67%, Escherichia coli was 100% resistance to

Augmentin and Ciprofloxacin indicating a high level of resistance in domestic sewage,

Klebsiella pneumonia exhibited 100% rsistance to Nalidixic acid and 66.67% resistance to

Ciprofloxacin. This study align with previous study Breathnach,Cubbon et al (2012), which

highlight the environmental contamination by multi drug resistance Pseudomonas aeruginosa,

in this current findings Pseudomonas aeruginosa was also the most prevalent multidrug resistant

both study underscore the environmental persistence and widespread nature of this pathogen.

Miranda et al (2015) This study reported that Pseudomonas aeruginosa, Escherichia coli and

Klebsiella pneumoniae shows resistance patterns with 12% against Ciprofloxacin and 100%

resistance against Augmentin, this study corroborates these findings particularly the 100%

resistance to Augmentin observed in Pseudomonas aeruginosa, However this present study 80%

resistance found in Escherichia coli compare to 12% reported by Miranda et al (2015). This

discrepancy might be due to difference in geographical locations, sampling periods, or local

antibiotic usage pattern. Ashbolt et al (2013). This study examined antibiotic resistance in

bacteria from various water sources and found significant levels of resistance, particularly in

urban wastewater. This present is consistent with Ashbolt et al (2013) findings showing high

level of resistance in bacteria isolated from both hospital effluents and domestic sewage, this

consistency suggests that wastewater sources are critical reservoirs of antibiotic resistant

bacteria.

Throughout this study, Pseudomonas aeruginosa, followed by Escherichia coli and Klebsiella

pneumoniae, were noted as the most significant Gram-negative bacteria exhibiting multidrug

resistance against almost all applied antibiotics. This was observed in both hospital effluent and
48
domestic sewage samples collected from the Dutsin-Ma metropolis.The high prevalence of P.

aeruginosa as a multidrug-resistant organism aligns with previous studies highlighting its ability

to survive in diverse environments and its significant role in environmental contamination,

(Breathnach, Cubbon, Karunaharan, Pope, & Planche, 2012). This persistence is likely due to the

organism’s intrinsic resistance mechanisms and its ability to acquire additional resistance genes

through horizontal gene transfer

This study reinforces the necessity for stringent monitoring and effective management of hospital

effluents and domestic sewage to mitigate the spread of multidrug-resistant bacteria.

Implementing advanced wastewater treatment technologies and enforcing policies to reduce

antibiotic use and discharge are crucial steps to combat the growing threat of AMR.

The observed resistance patterns in this study are consistent with those reported in other studies.

For instance, a study by Tacconelli et al. (2018) emphasized P.aeruginosa, E. coli, and K.

pneumoniae as critical priority pathogens due to their high resistance rates and significant impact

on public health. The resistance to Ciprofloxacin and Augmentin observed in this study aligns

with these findings, indicating a persistent issue of multidrug resistance in environmental and

clinical settings.

5.1 RECOMMENDATIONS
Based on the findings of this study on the antimicrobial susceptibility patterns of multidrug-

resistant Gram-negative bacteria from hospital effluent and domestic sewage in the Dutsin-Ma

metropolis, the following recommendations are proposed:

49
Enhance Wastewater Treatment:

Implement advanced wastewater treatment technologies capable of effectively removing

antibiotic residues and resistant bacteria. This includes options such as membrane bioreactors,

advanced oxidation processes, and constructed wetlands.Regularly monitor and upgrade existing

wastewater treatment facilities to ensure they meet the required standards for effectively

managing hospital effluents and domestic sewage.

Strengthen Antibiotic Stewardship Programs:

Promote the rational use of antibiotics in healthcare settings through robust antibiotic

stewardship programs. This includes monitoring antibiotic prescriptions, educating healthcare

professionals on appropriate antibiotic use, and reducing unnecessary antibiotic prescriptions.

Implement stringent policies to control the sale and use of antibiotics, particularly over-the-

counter sales, to prevent misuse and overuse.

Surveillance and Monitoring:

Establish continuous surveillance programs to monitor the prevalence and resistance patterns of

Gram-negative bacteria in hospital effluents and domestic sewage. This should include regular

sampling and testing to identify emerging resistance trends. Create a centralized database to

collect and analysed data on antimicrobial resistance (AMR) from various sources, facilitating

better understanding and management of AMR.

Infection Control Measures:

Strengthen infection control measures in healthcare facilities to reduce the spread of multidrug-

resistant bacteria. This includes strict adherence to hand hygiene, sterilization procedures, and

isolation protocols for patients infected with resistant organisms. Educate healthcare workers and

50
the public on the importance of infection control practices to prevent the transmission of resistant

bacteria.

Public Awareness and Education:

Conduct public awareness campaigns to educate the community about the dangers of antibiotic

misuse and the importance of proper sanitation and hygiene practices. Promote educational

programs in schools and community to raise awareness about AMR and encourage responsible

antibiotic use.

Research and Development:

Encourage research on alternative treatments and the development of new antibiotics to combat

multidrug-resistant bacteria. This includes funding for research on novel antimicrobial agents

and alternative therapies, such as bacteriophage therapy and antimicrobial peptides. Support

studies that explore the environmental impact of antibiotic residues and the effectiveness of

various wastewater treatment methods in removing these residues.

Policy and Regulation:

Implement and enforce regulations that limit the discharge of antibiotics and resistant bacteria

into the environment. This includes setting strict guidelines for pharmaceutical industries,

hospitals, and agricultural practices. Develop policies that promote sustainable agricultural

practices, such as the prudent use of antibiotics in livestock farming, to prevent the spread of

resistance from agricultural sources to human populations.

Collaboration and Partnerships:

Foster collaboration between healthcare providers, environmental agencies, policymakers, and

researchers to develop and implement comprehensive strategies to combat AMR. Participate in

51
global initiatives and partnerships aimed at addressing AMR, sharing data, and adopting best

practices from successful programs in other regions.

5.2 CONCLUSION
The study of antimicrobial susceptibility patterns of multidrug-resistant Gram-negative bacteria

from hospital effluent and domestic sewage in Dutsin-Ma metropolis reveals a significant

presence of resistant pathogens, particularly Pseudomonas aeruginosa, Escherichia coli, and

Klebsiella pneumoniae. These findings highlight the critical issue of antibiotic resistance, which

poses a serious threat to public health.

Pseudomonas aeruginosa exhibited high resistance rates to multiple antibiotics, including 100%

resistance to Augmentin and significant resistance to Nalidixic acid, Septrin, Gentamicin,

Ciprofloxacin, and Ceporex. Escherichia coli and Klebsiella pneumoniae also demonstrated

considerable resistance to a range of antibiotics, with high rates of resistance to Nalidixic acid,

Augmentin, and Ciprofloxacin. The study's results align with previous research, confirming the

widespread issue of Multidrug - resistant bacteria in both hospital effluent and domestic sewage.

The presence of multidrug-resistant bacteria in hospital effluent and domestic sewage indicates a

potential risk for the dissemination of these pathogens into the community and the environment,

exacerbating the public health threat posed by antimicrobial resistance (AMR).The high

resistance rates observed necessitate urgent action to implement effective measures to control

and mitigate the spread of these resistant bacteria.

52
 REFERENCE

American Public Health Association (2017). Standard Methods for the Examination of Water

and Wastewater. American Public Health Association.

Ashbolt, N. J. (2015). Microbial Contamination of Drinking Water and Human Health from

Community Water Systems. Current Environmental Health Reports, 2(1), 95–106.

Andersson, D. I., & Hughes, D. (2014). Microbiological effects of sublethal levels of antibiotics.

Nature Reviews Microbiology, 12(7), 465–478.

Allen, H. K., Donato, J., Wang, H. H., Cloud-Hansen, K. A., Davies, J., & Handelsman, J.

(2010). Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews

Microbiology, 8(4), 251–259.

Borchardt, M. A., & Spencer, S. K. (2019). Chapter 13 - Wastewater Pathogen Research and

Public Health Policy. In J. M. Li (Ed.), Microbial Source Tracking: Methods, Applications, and

Case Studies (pp. 333-349). Springer.

Baquero, F., & Martínez, J. L. (2017). Interventions on antibiotic resistance: Time for structural

changes. Science Translational Medicine, 9(417), eaal3653.

Blair, J. M., Webber, M. A., Baylay, A. J., Ogbolu, D. O., & Piddock, L. J. (2015). Molecular

mechanisms of antibiotic resistance. Nature Reviews Microbiology, 13(1), 42-51.

Bengtsson-Palme, J., Larsson, D. G. J., & Kristiansson, E. (2018). Using metagenomics to

investigate human and environmental resistomes. Journal of Antimicrobial Chemotherapy, 73(8),

2062-2070.

53
Baquero, F., Martínez, J.L., and Cantón, R. (2008). Antibiotics and antibiotic resistance in water

environments. Current Opinion in Biotechnology, 19(3), 260-265.

Berendonk, T.U., Manaia, C.M., Merlin, C., et al. (2015). Tackling antibiotic resistance: the

environmental framework. Nature Reviews Microbiology, 13(5), 310-317.

Breidenstein EB, de la Fuente-Núñez C, Hancock RE. Pseudomonas aeruginosa: all roads lead to

resistance. Trends Microbiol. 2011;19(8):419-426.

Czekalski, N., Berthold, T., Caucci, S., Egli, A., & Bürgmann, H. (2016). Increased levels of

multiresistant bacteria and resistance genes after wastewater treatment and their dissemination

into Lake Geneva, Switzerland. Frontiers in Microbiology, 7, 1626.

Clinical and Laboratory Standards Institute. (2020). Performance Standards for Antimicrobial

Susceptibility Testing. Clinical and Laboratory Standards Institute.

Davies, J., and Davies, D. (2010). Origins and evolution of antibiotic resistance. Microbiology

and Molecular Biology Reviews, 74(3), 417-433.

Forbes, B. A., Sahm, D. F., & Weissfeld, A. S. (2007). Bailey & Scott's Diagnostic Microbiology

(12th ed.). Mosby.

Centers for Disease Control and Prevention (CDC). (2019). Antibiotic Resistance Threats in the

united States, 2019. Retrieved from https://www.cdc.gov/drugresistance/pdf/threats-report/2019-

ar-threats-report-508.pdf

Fernando, D. M., Tun, H. M., Poole, J., Patidar, R., Li, R., Mi, R., & Khafipour, E. (2020).

Detection of antibiotic resistance genes in source and drinking water samples from a first nation

community in Canada. Applied and environmental microbiology, 86(1), e01700-19.

54
Gaze, W. H., Zhang, L., Abdouslam, N. A., Hawkey, P. M., Calvo-Bado, L., Royle, J.,&

Wellington, E. M. (2011). Impacts of anthropogenic activity on the ecology of class 1 integrons

and integron-associated genes in the environment. The ISME journal, 5(8), 1253-1261.

Gaze, W.H., Krone, S.M., Larsson, D.G.J., et al. (2013). Influence of humans on evolution and

mobilization of environmental antibiotic resistome. Emerging Infectious Diseases, 19(7),

e120871.

Gellatly SL, Hancock RE. Pseudomonas aeruginosa: new insights into pathogenesis and host

defenses . Pathog Dis. 2013; 67 (3):159-173.

Larsson, D. G. J., de Pedro, C., & Paxeus, N. (2007). Effluent from drug manufactures contains

extremely high levels of pharmaceuticals. Journal of hazardous materials, 148(3), 751-755.

Leekha, S., Terrell, C. L., & Edson, R. S. (2011). General principles of antimicrobial therapy.

Mayo Clinic Proceedings, 86(2), 156-167.

Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A. K. M., Wertheim, H. F. L., Sumpradit, N.,&

Greko, C. (2013). Antibiotic resistance-the need for global solutions. The Lancet Infectious

Diseases, 13(12), 1057-1098.

Lister PD, Wolter DJ, Hanson ND. Antibacterial-resistant Pseudomonas aeruginosa: clinical

impact and complex regulation of chromosomally encoded resistance mechanisms. Clin

Microbiol Rev. 2009;22(4):582-610.

Manaia, C. M., Rocha, J., Scaccia, N., Marano, R., Radu, E., Biancullo, F., ... & Vaz-Moreira, I.

(2018). Antibiotic resistance in wastewater treatment plants: tackling the black box. Environment

international, 115, 312-324.

Mara, D., & Horan, N. (2003). Handbook of Water and Wastewater Microbiology. Academic

Press.
55
Martinez, J.L. (2008). Antibiotics and antibiotic resistance genes in natural environments.

Science, 321(5887), 365-367.

Michael, I., Rizzo, L., McArdell, C.S., et al. (2013). Urban wastewater treatment plants as

hotspots for the release of antibiotics in the environment: a review. Water Research, 47(3), 957-

995.

Oliver A, Mulet X, López-Causapé C, Juan C. The increasing threat of Pseudomonas aeruginosa

high-risk clones. Drug Resist Updat. 2015;21-22:41-59.

Prescott, L. M., Harley, J. P., & Klein, D. A. (2005). Microbiology (6th ed.). McGraw-Hill

Education.

Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy,

typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998;11(4):589-603.

Paczosa MK, Mecsas J. Klebsiella pneumoniae: Going on the Offense with a Strong Defense.

Microbiol Mol Biol Rev. 2016;80(3):629-661.

Ramirez MS, Traglia GM, Lin DL, et al. Klebsiella pneumoniae carbapenemase, Australia.

Emerg Infect Dis. 2014;20(5):896-897.

Spellberg, B., Blaser, M., Guidos, R. J., Boucher, H. W., Bradley, J. S., Eisenstein, B. I., ... &

Gilbert, D. N. (2011). Combating antimicrobial resistance: policy recommendations to save lives.

Clinical Infectious Diseases, 52(suppl_5), S397-S428.

Shon AS, Bajwa RP, Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a

new and dangerous breed. Virulence. 2013;4(2):107-118.

Tacconelli, E., Carrara, E., Savoldi, A., Harbarth, S., Mendelson, M., Monnet, D. L., ... &

Holmes, A. H. (2018). Discovery, research, and development of new antibiotics: the WHO

56
priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet Infectious Diseases,

18(3), 318-327.

Theuretzbacher, U. (2017). Global antibacterial resistance: the never-ending story. Journal of

Global Antimicrobial Resistance, 8, 1-2.

Tchobanoglous, G., Burton, F. L., & Stensel, H. D. (2003). Wastewater Engineering: Treatment

and Reuse (4th ed.). McGraw-Hill.

United States Environmental Protection Agency. (2020). Clean Water Act. Retrieved from

https://www.epa.gov/laws-regulations/summary-clean-water-act.

United Nations. (2018). World Water Development Report 2018: Nature-Based Solutions for

Water. UNESCO.

Ventola, C. L. (2015). The antibiotic resistance crisis: part 1: causes and threats. Pharmacy and

Therapeutics, 40(4), 277-283.

World Health Organization. (2019). Guidelines for Drinking-water Quality (4th ed.). World

Health Organization.

World Health Organization. (2006). Guidelines for the Safe Use of Wastewater, Excreta, and

Greywater: Volume 2: Wastewater Use in Agriculture. World Health Organization.

Wright, G. D. (2010). Antibiotic resistance in the environment: a link to the clinic? Current

Opinion in Microbiology, 13(5), 589–594.

Wellington, E. M., Boxall, A. B., Cross, P., Feil, E. J., Gaze, W. H., Hawkey, P. M., ... &

Johnson-Rollings, A. S. (2013). The role of the natural environment in the emergence of

antibiotic resistance in gram-negative bacteria. The Lancet Infectious Diseases, 13(2), 155–165.

57
Yang, Y., Li, B., Zou, S., Fang, H. H. P., Zhang, T., & Li, X. (2017). Fate of antibiotic resistance

genes in sewage treatment plant revealed by metagenomic approach. Water research, 124, 419-

430.

Baquero, F., Martínez, J.L., and Cantón, R. (2008). Antibiotics and antibiotic resistance in water

environments. Current Opinion in Biotechnology, 19(3), 260-265.

Bradford, P.A. (2001). Extended-spectrum β-lactamases in the 21st century: characterization,

epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews,

14(4), 933-951.

Dyar, O.J., Huttner, B., Schouten, J., and Pulcini, C. (2017). What is antimicrobial stewardship?

Clinical Microbiology and Infection, 23(11), 793-798.

Kümmerer, K. (2009). Antibiotics in the aquatic environment–a review–part I. Chemosphere,

75(4), 417-434.

Lister, P.D., Wolter, D.J., and Hanson, N.D. (2009). Antibacterial-resistant Pseudomonas

aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance

mechanisms. Clinical Microbiology Reviews, 22(4), 582-610.

Martínez, J.L., Baquero, F., and Andersson, D.I. (2007). Predicting antibiotic resistance. Nature

Reviews Microbiology, 5(12), 958-965.

Nordmann, P., Cuzon, G., and Naas, T. (2011). The real threat of Klebsiella pneumoniae

carbapenemase- producing bacteria. The Lancet Infectious Diseases, 11(9), 683-686.

Novo, A., Manaia, C.M., and Martins da Costa, P. (2013). The impact of domestic sewage on the

prevalence of antibiotic-resistant bacteria in aquatic environments. Journal of Environmental

Monitoring, 15(1), 93-98.

58
Paterson, D.L., and Bonomo, R.A. (2005). Extended-spectrum β-lactamases: a clinical update.

Clinical Microbiology Reviews, 18(4), 657-686.

Pitout, J.D., Nordmann, P., and Poirel, L. (2015). Carbapenemase-producing Klebsiella

pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrobial Agents and

Chemotherapy, 59(10), 5873-5884.

Verlicchi, P., Al Aukidy, M., and Zambello, E. (2010). Occurrence of pharmaceutical

compounds in urban wastewater: removal, mass load and environmental risk after a secondary

treatment—a review. Science of the Total Environment, 429, 123-155.

WHO (2014). Antimicrobial resistance: global report on surveillance. World Health

Organization.

59