CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of Study

The detection of Carbapenemase producers among Enterobacteriaceae (CPE) is an

important area of research in the field of Microbiology and clinical medicine. With

a clear endemicity according to enzyme type, carbapenemase-producing

enterobacteriaceae (CPE) have already been identified all over the world. The most

common situations in Europe have been reported in Greece, where 60.5% of

Klebsiella pneumoniae (a total of 1460 isolates) are carbapenem-resistant, and in

Italy, where 28.8% of those isolates are carbapenem-resistant (a total of 841

isolates) during the 2012 European Antimicrobial Resistance Surveillance Network

(EARS-Net) project (EARS Report 2012). It is unknown how common CPE occurs

in the community, however it is likely heterogeneous, as seen in the prevalence of

extended-spectrum β-lactamases (ESBL-producing) Escherichia coli; 3-5% in

France; >80% in India (Nordmannet al., 2011).

The Enterobacteriaceae family, which comprises genera and species that cause

both nosocomial infections and clearly characterized illnesses, is the most

significant family of bacteria in human medicine. The members of this family are

Gram-negative, rod-shaped, non-spore-forming facultative anaerobes that ferment

1
glucose and other sugars, reduce nitrate to nitrite, and produce catalase but seldom

oxidase.

Escherichia coli (E. coli) and Klebsiella pneumoniae are two well-known gram-

negative pathogens that belong to the family Enterobacteriaceae. Other important

genera include; salmonella, shigella, serratia etc. These organisms are typically

present in the gut and have the potential to infect many other organ systems,

including the urinary tract, circulation, respiratory system, and intra-abdominal

cavity, resulting to sporadic infections and outbreaks (David, 2006). Treating

infections caused by Enterobacteriaceae especially Klebsiella becomes more

difficult due to their developing resistance to several antibiotic classes.

Carbapenemases are β- lactamase enzymes produced by certain bacteria capable of

breaking down and inactivating the carbapenem antibiotics, which are considered

the last line of defense against multidrug-resistant infections. The most recent β-

lactam antibiotics on the market, carbapenems, including imipenem/cilastatin,

meropenem, doripenem, and ertapenem. They have a broad spectrum of activity

and are typically used to treat infections caused by multidrug-resistant (MDR)

pathogens.

The selection of antibiotics for treating infections caused by Gram-negative

bacteria is severely constrained, due to the formation of acquired carbapenemases.

Multidrug resistance, including bacteria resistant to all current antibiotics, is

2
caused by the horizontal transmission of carbapenamase genes through mobile

genetic elements, carrying additional resistance elements that give resistance to

multiple groups of antibiotics. (Kumarasamyet al., 2010).

Typically, screening for the presence of carbapenemase genes in clinical isolates is

used in studies to identify carbapenemase producers among Enterobacteriaceae.

This involves the use molecular methods like DNA sequencing or PCR to pinpoint

specific genes linked to carbapenem resistance. Moreover, carbapenemase

enzymes can be found using phenotypic techniques like the Modified Hodge Test

or Carba NP Test.

1.2 Significance of the Study

Carbapenemase-producing Enterobacteriaceae (CPE) are a major public health

concern due to their resistance to the carbapenems, which are a class of last-line

antibiotics. As a result, infections caused by CPE can be fatal and challenging to

cure. This study therefore aims at investigating rapid and accurate methods for

detecting Carbapenemase-producing Enterobacteriaceae (CPE) in clinical samples,

which is crucial in combating antibiotic resistance, implementing infection control

measures to prevent the spread of these organism as well as informing public

health strategies.

3
1.3 Aim of Study

This study aims to examine and characterize bacteria belonging to the

Enterobacteriaceae family, including Escherichia coli and Klebsiella that are

capable of producing the enzymes known as carbapenemases, which degrade and

render carbapenem antibiotics inactive.

1.4 Objectives of the Study

The specific objectives are:

i. To evaluate the antibacterial activities of Meropenem against Klebsiella and

Escherichia coli species.

ii. To perform antibiotic susceptibility test using the Disk diffusion method, to

identify potential carbapenemase producers within the collected isolates.

iii. To interpret the MEM disc zones of inhibition using the Clinical and

Laboratory Standards Institute (CLSI) standard.

4
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Antibiotics

Antibiotics are a class of pharmaceutical agents produced wholly or partially by

chemical synthesis, that are used to treat bacterial infections. They were initially

coined from the word ‘antibiosis’ which indicates ‘against life’. They either inhibit

the growth of bacteria or kill them, in low concentrations (Zaffiri, Gardner, and

Toledo-Pereyra, 2012). These drugs work by targeting specific mechanisms within

bacteria, such as cell wall synthesis, protein synthesis, nucleic acid synthesis, and

metabolic pathways (Davies and Davies, 2010). By inhibiting these essential

bacterial processes, antibiotics effectively combat infections and aid in the

recovery of affected individuals.

The discovery of antibiotics in the 20th century marked a turning point in

healthcare, significantly reducing mortality and morbidity associated with bacterial

infections (Aminov, 2019). The story began with the fortuitous observation of

penicillin's antibacterial properties by Alexander Fleming in 1928 (Fleming, 1929).

This discovery paved the way for the development of countless other antibiotics,

each with unique characteristics and applications.

5
Several classes of antibiotics exist, including penicillins, cephalosporins,

tetracyclines, aminoglycosides, fluoroquinolones, and others (Katzung, Masters,

and Trevor, 2017). Each class exhibits unique mechanisms of action and is

effective against specific types of bacteria. The overuse and misuse of antibiotics

have led to concerns regarding antibiotic resistance, prompting efforts to promote

judicious antibiotic prescribing and stewardship (World Health Organization,

2018).

2.2 Carbapenems

Carbapenems are a class of β-lactam antibiotics with a broad spectrum of

antimicrobial activity, including activity against multidrug-resistant bacteria such

as extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and

Pseudomonas aeruginosa (Paterson and Harris, 2015). These antibiotics are

structurally similar to penicillins and cephalosporins, but they possess a broad

spectrum of activity and are more resistant to β-lactamase enzymes (Livermore,

2009).

The Carbapenems belong to the β-lactam class, which also includes carbapenems,

monobactams, cephalosporins and penicillins and compared to most other beta

lactam antibiotics, this class of antimicrobial is effective against both gram positive

and gram negative bacteria having a broader spectrum of activity.

6
Carbapenems exert their antibacterial effects by inhibiting cell wall synthesis in

bacteria, ultimately leading to cell lysis and death (Drawz and Bonomo, 2010).

They are considered as one of the last-line treatment options for serious infections

and are often reserved for use in healthcare settings to minimize the development

of resistance (Papp-Wallace et al., 2011).

In recent years, the emergence of Carbapenem-resistant Enterobacteriaceae (CRE)

has raised serious public health concerns, highlighting the importance of judicious

use and stewardship of these critical antibiotics (Logan and Weinstein, 2017).

7
Figure 1: Carbapenem Backbone
Source: www.microbenotes.com/carbapenems/

8
2.3 Carbapenemases

Carbapenemases are enzymes produced by certain bacteria that confer resistance to

carbapenem antibiotics. They are a major concern in healthcare settings due to

their ability to hydrolyze and inactivate carbapenems, which are considered to be

the last resort treatment for multidrug-resistant infections, rendering these

antibiotics ineffective against the bacteria (Paterson and Harris, 2015).

Carbapenemases are a type of β-lactamase, which is a class of enzymes that can

break down the β-lactam ring present in many β-lactam antibiotics, including

carbapenems (Drawz and Bonomo, 2010).

Carbapenemases are classified into different molecular classes, with each class

identified by their unique genetic sequences and characteristics, including class A

(e.g., KPC), class B (e.g., IMP, VIM, NDM), class D (e.g., OXA-48), and some

others (Nordmann, Dortet, and Poirel, 2012). These classes of carbapenemases

have been associated with different bacterial species and are responsible for the

spread of carbapenem resistance worldwide.

2.3.1 Class A Carbapenemases

Class A carbapenemases (serine β-lactamases) are a group of β-lactamase

enzymes, that are primarily found in Enterobacterales, such as Klebsiella

pneumoniae and Escherichia coli, that are capable of hydrolyzing carbapenem

9
antibiotics. They are also commonly gram negative associated and confer a high

level of resistance to carbapenems. One of the most well-known class A

carbapenemases is KPC (Klebsiella pneumoniae carbapenemase), which has been

associated with significant outbreaks of carbapenem-resistant Enterobacteriaceae

(Paterson and Harris, 2015).

KPC enzymes are encoded by genes located on plasmids, which allows for easy

transfer between different bacterial species and contributes to the spread of

carbapenem resistance (Drawz and Bonomo, 2010). The detection of KPC-

producing bacteria is important for infection control and treatment decisions in

healthcare settings.

2.3.2 Class B Carbapenemases

Class B carbapenemases, also known as metallo-β-lactamases (MBLs), are a type

of carbapenemase that can hydrolyze carbapenem antibiotics by utilizing zinc ions

in their active sites. These enzymes are of particular concern due to their ability to

confer resistance to carbapenems and other β-lactam antibiotics, with few effective

treatment options available. One of the most clinically significant MBLs is the

New Delhi metallo-β-lactamase (NDM), which has been associated with antibiotic

resistance in various Gram-negative bacteria, such as Klebsiella pneumoniae and

Escherichia coli (Pitout and Wieczorkiewicz, 2009).

10
MBLs are often plasmid-mediated, allowing for their rapid spread among bacterial

populations and contributing to the global dissemination of carbapenem resistance

(Nordmann et al., 2012). The emergence of MBL-producing bacteria presents a

significant challenge in clinical practice, requiring robust surveillance and

infection control measures to prevent their spread and minimize the impact of

multidrug-resistant infections.

2.3.3 Class C Carbapenemases

Class C carbapenemases, also known as AmpC β-lactamases, are a type of

carbapenemase that can hydrolyze carbapenem antibiotics, leading to resistance in

Gram-negative bacteria. They are often plasmid-mediated, allowing for their

spread among bacterial populations and contributing to the global dissemination of

carbapenem resistance (Papp-Wallace et al., 2011). Class C carbapenemases are

particularly concerning because they have the potential to confer resistance not

only to carbapenems but also to other β-lactam antibiotics, further limiting

treatment options for infections caused by resistant bacteria.

One of the most clinically significant Class C carbapenemases is the plasmid-

mediated AmpC β-lactamase, which has been associated with resistance in various

Gram-negative bacteria, such as Enterobacter spp. and Pseudomonas aeruginosa

(Jacoby et al., 2009). The emergence and spread of Class C carbapenemases

11
present a significant challenge in clinical practice, underscoring the importance of

surveillance and infection control measures to prevent their dissemination and

minimize the impact of multidrug-resistant infections.

2.3.4 Class D Carbapenemases

Class D carbapenemases, also known as oxacillinases, are a type of carbapenemase

that confer resistance to carbapenem antibiotics in Gram-negative bacteria. They

are characterized by their ability to hydrolyze carbapenems and other β-lactam

antibiotics, contributing to multidrug resistance in clinically important pathogens

such as Acinetobacter baumannii and Pseudomonas aeruginosa (Drawz and

Bonomo, 2010). Class D carbapenemases are often encoded by genes located on

mobile genetic elements, facilitating their transfer between bacteria and

contributing to the dissemination of carbapenem resistance in healthcare settings.

One of the most well-known Class D carbapenemases is the OXA-type enzymes,

which have been associated with outbreaks of carbapenem-resistant Acinetobacter

baumannii infections in healthcare settings worldwide (Evans and Amyes, 2014).

The emergence of Class D carbapenemases underscores the urgent need for

surveillance, infection control, and antimicrobial stewardship to mitigate the spread

of carbapenem-resistant bacteria and preserve the effectiveness of available

treatment options.

12
2.4 Enterobacteriaceae

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

numerous genera, such as Escherichia, Salmonella, Klebsiella, and Enterobacter.

The members of this family are gram negative, rod-shaped, non-spore forming,

facultative anaerobes that ferment glucose and other sugars, reduce nitrate to

nitrites and produce catalase but seldom oxidase.

These bacteria are commonly found in the gastrointestinal tract of humans and

animals, and some species can cause a wide range of infections, including urinary

tract infections, bloodstream infections, and gastrointestinal infections (Podschun

and Ullmann, 1998).

Enterobacteriaceae are known for their ability to develop resistance to multiple

antibiotics through mechanisms such as extended-spectrum β-lactamases (ESBLs)

and carbapenemases, posing a significant challenge to the treatment of infections

caused by these organisms (Paterson and Bonomo, 2005).

2.4.1 Klebsiella

Klebsiella is a genus of Gram-negative, non-motile, facultative anaerobic bacteria

within the family Enterobacteriaceae. Some species of Klebsiella, such as

Klebsiella pneumoniae and Klebsiella oxytoca, are known for their ability to cause

13
a range of infections in humans, including urinary tract infections, pneumonia, and

bloodstream infections (Podschun and Ullmann, 1998).

Klebsiella pneumoniae is particularly concerning due to its association with

healthcare-associated infections and its ability to acquire resistance to multiple

antibiotics, including carbapenems. This has led to the emergence of carbapenem-

resistant Klebsiella pneumoniae (CRKP), which poses a significant challenge for

the treatment of infections caused by this pathogen (Paterson and Bonomo, 2005).

In addition to antibiotic resistance, some strains of Klebsiella have been shown to

produce extended-spectrum beta-lactamases (ESBLs), which further limits the

choice of effective antimicrobial therapy. Due to its ability to acquire and spread

antibiotic resistance determinants, Klebsiella pneumoniae has become a significant

challenge in clinical practice, leading to increased mortality and healthcare costs

(Navon-Venezia et al., 2017).

14
Scientific Classification

Domain: Bacteria

Phylum: Pseudomonadota

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Klebsiella

Species: Klebsiella pneumonia

Klebsiella aerugenes

Klebsiella granulomatis

Klebsiella oxytoca

Klebsiella milletis

Klebsiella kielensis

Klebsiella grimontii

Klebsiella huaxiensis

15
2.4.2 Escherichia coli

Escherichia coli, commonly known as E. coli, is a Gram-negative, facultative

anaerobic bacterium belonging to the family Enterobacteriaceae (Rasko and

Sperandio, 2010). It is a versatile microorganism prevalent in the environment and

is a normal part of the intestinal microbiota in humans and animals (Rasko and

Sperandio, 2010). While most strains of E. coli are harmless, some pathogenic

strains can cause a range of illnesses, including urinary tract infections,

gastroenteritis, and even life-threatening diseases such as hemolytic uremic

syndrome (Kaper, Nataro, and Mobley, 2004).

E. coli strains are classified into different pathotypes based on their virulence

factors and disease manifestations. For example, enterotoxigenic E. coli (ETEC) is

known for causing traveler's diarrhea, while enterohemorrhagic E. coli (EHEC) is

associated with foodborne illness outbreaks (Kaper, Nataro, and Mobley, 2004).

Furthermore, some strains of E. coli have acquired resistance to multiple

antibiotics, posing a significant public health concern (Tadesse and Zhao, 2012).

The diverse pathogenicity and antibiotic resistance profiles of E. coli make it a

clinically significant bacterium that requires ongoing surveillance and research

efforts to mitigate its impact on public health (Tadesse and Zhao, 2012).

16
Scientific Classification

Domain: Bacteria

Phylum: Pseudomonadota

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Escherichia

Species: Escherichia coli

17
2.5 Epidemiology and Global Distribution of Carbapenemase Producers

The widespread distribution of Carbapenem-Resistant Enterobacteriaceae (CRE) is

mainly due to their synthesis of carbapenemases and the horizontal transfer of the

producing genes via plasmids. The prevalence of CRE and the carbapenemase

species involved are highly dependent upon the geographic region. Three groups of

carbapenemases; Klebsiella Pneumonia Carbapenemase (KPC), New Delhi

Metallo-beta-lactamase (NDM), and Oxacillin-48 (OXA-48) are currently

considered to be the three major β-lactamases of epidemiological and clinical

significance.

A Klebsiella pneumoniae (KPN) strain with a plasmid-mediated carbapenemase

gene generating a protein later known as K. pneumonia carbapenemase (KPC) was

initially identified in the United States in 2001 (Yigit et al., 2001). Since then,

blaKPC have expanded significantly throughout South America and the United

States. Additionally, outbreaks of Enterobacteriaceae that produce KPC have been

documented in most of Europe in sequential order (Patel and Bonomo, 2013).

The BlaKPC–2 has been the most extensively distributed carbapenemase gene

since the first KPC-producing CRE strain was discovered in 2017, China (Wei et

al., 2007, Zhang et al., 2017). The primary KPC that produced clinically isolated

CRE was KPN. According to multilocus sequence typing (MLST) of the majority

18
of KPC-producing KPN strains, clonal complex 258 (CC258) acquired a KPC-

encoding gene during the early CRE epidemic and quickly proliferated (Bowers et

al., 2015). Sequence type (ST) ST11 is most common in China, ST258 is most

common in the US, while ST340, ST437, and ST512 are most common in other

nations (Chen et al., 2014). Thus, the primary method thought to be responsible for

the dissemination of KPC-producing KPN is clonal transmission.

The first reports of blaNDM-associated carbapenem-resistant Klebsiella

pneumonia (KPN) were made in India in 2009 (Yong et al., 2009). Since then, the

majority of Enterobacteriaceae species have been found to have blaNDM. Dortet et

al. (2014) states that NDM-type β-lactamase is primarily found across Asia,

specifically in China, India, Pakistan, and Bangladesh. New Delhi Metallo-beta-

lactamase-mediated carbapenemase (NDM) is now the second most common

carbapenemase among Carbapenemase-Resistant Enterobacteriaceae (CRE) in

China, but blaNDM is more common in Escherichia coli (Zhang et al., 2018). A

significant diversity of blaNDM-associated E. coli has been found as a result of the

horizontal transmission of pandemic broad-host-range plasmids, with ST131,

ST167, and ST410 being the predominant kinds. In addition, blaIMP have

proliferated over Japan (Pitout et al., 2015).

Greece is the epicenter of Enterobacteriaceae that produce Pseudomonas

aeruginosa (VIM). Other European countries like the United Kingdom, Belgium,
19
Spain, Italy, and Hungary undoubtedly have serious outbreaks, as do certain Asian

nations like Taiwan, China, and South Korea (Walsh et al., 2005).

The oxacillinases (OXA) are class D β-lactamases that work by breaking oxacillin.

The first OXA-encoding gene, known as blaOXA–23, was discovered in an

Acinetobacter baumannii strain from the United Kingdom in 1985 (Donald et al.,

2000). Since then, other members of the OXA family OXA-23-like, OXA-48-like,

OXA-40-like, OXA-51-like, and OXA-58-like have been progressively identified

in the Enterobacteriaceae. OXA-48, the most prevalent class D β-lactamase, was

initially discovered in a KPN isolate from Turkey in 2001 (Evans and Amyes,

2014).

20
Plate 1: The Global distribution of different Carbapenemases in CPE
Source: www.frontiersin.org

21
2.6 Diagnostic Methods for Detecting Carbapenamase Producers

Carbapenamase-resistant Enterobacteriaceae (CRE) infections represent a serious

risk to public health due to its resistance to β-lactams, aminoglycosides, and

fluoroquinolones. Nosocomial and community-acquired infections are starting to

fail therapy because of CRE (Gautier et al., 2018). As a result, the identification of

isolates that produce carbapenemase must be done quickly and accurately.

As per the Clinical and Laboratory Standards Institute (CLSI) criteria, isolates of

Enterobacteriaceae are considered to be potential makers of carbapenemase if the

minimum inhibitory concentrations (MICs) of imipenem or meropenem are 2–

4μg/ml or 2 μg/ml for ertapenem (Weinstein MP, 2018). However, some

carbapenemase-producing bacteria might show lower MICs for carbapenems than

the clinical breakpoints proposed by the Clinical and Laboratory Standards

Institute (CLSI). Currently, the two primary approaches for detecting

carbapenemasesfrom cultured isolates are molecular-based methods and

phenotypic detection assays.

2.6.1 Phenotypic Detection Assays

The Modified Hodge test (MHT) is a common phenotypic method for the detection

of CPE. It determines if the test strain has an inactivation effect on antibacterial

medications based on whether the growth of the indicator strain is enhanced at the

22
intersection of the growth line and the inhibition zone created by the indicator

strain and the test strain, respectively (Girlich et al., 2012). This approach has a

low sensitivity (<50%) for detecting class B β-lactamases, but a high specificity

and sensitivity for identifying KPC-producing CRE. The Triton Hodge test, also

known as Triton X-100, was presented as a way to get around this restriction. This

technique enhanced the sensitivity of simultaneously detecting other

carbapenemases and raised the identification of NDM-producing clinical isolates to

>90% (Pasteran et al., 2016).

The Carba NP test is a colorimetric assay that was later developed by Nordmann. It

is faster than MHT and has a lower false-positive rate (Nordmann et al., (2012). In

this test, the simultaneous change in the color of phenol red, which is subjectively

assessed by the laboratory operator, is monitored along with the change in pH of

the reaction system brought on by the carbapenemase hydrolysis of imipenem.

Moreover, this method could preliminarily identify carbapenemases types based on

tazobactam and EDTA (Dortet et al., 2012). Then, while developing the Blue-

Carba test, Pires et al. (2013) substituted bromothymol blue for phenol red as the

pH indicator, increasing the modified assay's sensitivity from 93.3 to 100%

(Novais et al., 2015).

23
2.6.2 Molecular-based Detection Assays

The most reliable methods for identifying carbapenemase genes are tests that rely

on molecular methods (Nordmann et al., 2011). These tests are able to identify the

carbapenemase precisely as well as whether the enzyme(s) is/are present. The

simplex and complex Polymerase Chain Reaction (PCR) assay is the most widely

utilized conventional molecular genotyping technique. However, it takes a lot of

time to identify a single gene using the conventional PCR method. Thus, time-

saving multiple PCR with excellent sensitivity and specificity was created

(Ellington et al., 2016).

Multiplex real-time PCR techniques were first developed between 2006 and 2012

in order to quickly detect the majority of carbapenemases, including KPC, OXA-

48, VIM, IMP and NDM (Monteiro et al., 2012). Additionally, a number of

modified techniques were put forth in an attempt to address the inaccuracy brought

about by the diversity of OXA-48-like carbapenemases. These techniques included

a multiplex PCR assay that employed peptide-nucleic acid probes to identify

resistance genes in a mixture of Enterobacteriaceae isolates with a high degree of

efficiency, as well as a real-time PCR assay based on a high-resolution melt

analysis (Hemarajata et al., 2015).

24
Table 1: Phenotypic tests for Carbapenamase detection in clinical isolates.

Test parameters Modified Hodge test Carba NP test

(MHT)

Carbapenem- Enterobacteriaceae Enterobacteriaceae, Pseudomona

resistant s aeruginosa
organisms

sensitivity, ∼95% and sensitivity, ∼84% and specificity,

Accuracy For Enterobacteriaceae, For Enterobacteriaceae,

specificity, ∼91%; ∼100%; for P. aeruginosa,

false-positive results sensitivity, 98% and specificity,
withESBL- or AmpC- 98%; with change in lysis buffer
positive isolates with and starting pH, sensitivity
porin defects increases to close to 99%
Ease of use Easy to perform, no For manual versions, fresh
special reagents or reagents need to be prepared
media necessary frequently due to limited shelf-
life of imipenem solution; pH
meter needed
Interpretation of Indentation of zone Color change from red to yellow
results diam of E.coli toward or blue to yellow depending on
carbapenem disk along the pH indicator; color change
streaked isolate; reading can be subjective for intermediate
results
of zone indentation can
be subjective
Total time to 15 min for setup and 2 40 to 170 min (varies based on
perform test min to read the test)
following day

Source: https://journals.asm.org/

25
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Area

The study was conducted in the University of Abuja Microbiology Laboratory,

Airport Road, Gwagwalada Area Council, Federal Capital Territory, Abuja. The

University of Abuja Main Campus is situated along Abuja-Airport Road, few

kilometres from the Nnamdi Azikwe International Airport. It lies between Latitude

8 °58’ N of the equator and Longitude 7 °10’ Eof the Greenwich meridian. It has a

land mass of approximately 118sq km making it one of the top ten Universities

with largest land mass in Nigeria. The University Main Campus is bounded in the

North by Anagada village, on the West by Giri, on the East and South East by

Kuje, and on the South-West by Gwagwalada town (UniAbuja Post Graduate

Handbook, 2015). The Territory is made up of 6 Area Councils; Abuja Municipal

Area Council, Gwagwalada, Kuje, Bwari and Kwali Area Council. At the 2006

census, the city of Abuja had a population 776,298 making it one of the ten most

populous cities in Nigeria (NPC, 2006).

3.2 Materials

The materials used for collection of samples include; sterile hand gloves, and

sterile petri plate. Other materials include; pipettes, inoculating wire loop, bunsen
26
burner, aluminum foil, cotton wool and syringes. Glassware used includes; conical

flask, measuring cylinder, glass rod and beaker.

Equipment used in the laboratory include; the autoclave, hot air oven, incubator,

refrigerator, weighing balance, and microscope.

The media used in the study include; Nutrient agar, Mueller-Hinton agar.

3.3 Collection of Samples

A total of twenty (20) clinical specimens of Escherichia coli and Klebsiella species

were collected in sterile nutrient agar plates from patients of the University of

Abuja Teaching Hospital (UATH) and transported to the University of Abuja

Microbiology laboratory for microbiological analysis.

3.4 Preparation of Media and Sterilization of Glassware

Mueller Hinton (MH) agar and nutrient agar plates were prepared according to the

manufacturer’s instructions. All glasswares were washed and sterilized in a hot air

oven for 2hours at 160°c.

Antibiotics used were Gentamicin (10 μg), Chloramphenicol (10 μg), Tetracycline

(50 μg), Ceftriaxone (10 μg), and Ciprofloxacin (5 μg) (CLSI, 2020).

3.5 Reconfirmation of Test Organisms

27
The collected strains were initially seeded on Nutrient agar using the quadrant

streaking method to obtain isolated colonies and only pure samples were

considered for the Disk diffusion test. Mueller Hinton (MH) agar is the differential

media used for growing isolated colonies of Klebsiella and Escherichia coli

species (Betty et al., 2007).

3.6 Disk Diffusion Test

The antibiotic susceptibility testing of each isolate was performed on Mueller

Hinton (MH) agar using the Disk diffusion method. A few colonies of Klebsiella

and Escherichia coli were picked from the pure culture and suspended in sterile

water to give a density equivalent to 0.5 McFarland standard and then inoculated

onto the Mueller Hinton (MH) agar plates.

All procedures were done using Kirby Bauer disk diffusion method. The

Ceftrizone disc alongside four (4) other antibiotic discs; Tetracycline, Gentamicin,

Ciprofloxacin and Chloramphenicol, were placed firmly forming a ring on the agar

plate. The plates were inverted and incubated at 37°c for 24hours. After 24hours,

the petri plates were taken out, observation made and the zones of inhibition

measured.

28
3.6.1 Data Collection

The isolates were labelled E1-10 and K1-10 to represent Escherichia coli and

Klebsiella spp. respectively.

3.6.2 Interpretation of Results

For IPM, ETP and MEM disc zones of inhibition, ≥ 23mm indicates sensitivity, 20

to 22mm indicates resistance (CLSI, 2010).

3.7 Data Analysis

The data collected were reported using tables, while comparison was done using

descriptive statistics graphs.

29
 CHAPTER FOUR

4.0 RESULTS

4.1 Evaluation of Antimicrobial Activities of Ceftrixone against Klebsiella

spp. and Escherichia coli

The produced antibiotics Ceftriaxone’s action on the sampled organisms

(Klebsiella spp. and Escherichia coli) is shown in Table 2 and 3, together with the

diameter zones of inhibition measured in millimetres (mm), showing a region of

clearance, suggesting the organisms produced the ESBL which inhibit some ESBL

class of antibiotics.

30
 Antibiotic Klebsiella spp. Samples Zone of Inhibition(mm)

Ceftriaxone K1 2.1

K2 1.7

K3 1.4

K4 1.9

K5 1.2

K6 1.1

K7 1.0

K8 Not Clear

K9 Not Clear

K10 Not Clear

Table 2: Zone of Inhibition (mm) of Klebsiella spp. against Ceftriaxone

Key: K1 - K10 = Klebsiella spp. samples

31
Table 3: Zone of Inhibition (mm) of Ceftriaxone against Escherichia coli

Antibiotic Escherichia coli Samples Zone of Inhibition (mm)

Ceftriaxone E1 1.4

E2 1.0

E3 1.5

E4 2.1

E5 2.0

E6 1.7

E7 1.2

E8 1.5

E9 2.8

E10 2.2

Key: E1 - E10 = Escherichia coli isolates

32
A comparison of four (4) other antibiotic against Klebsiella spp. and Escherichia

coli.

33
Table 4: Zone of Inhibition (mm) of other Antibiotics against Klebsiella spp.

Klebsiella spp. Antibiotics Zone of Inhibition (mm)

Samples

T G CI CH

K1 22 28 23 14

K2 10 20 22 12

K3 15 22 15 23

K4 13 24 09 22

K5 15 27 12 21

K6 15 26 11 22

K7 12 22 12 21

K8 19 18 13 22

K9 12 21 14 22

K10 11 23 14 23

Keys:
T= Tetracycline CI= Ciprofloxacin
G= Gentamicin CH= Chloramphenicol
K1-10 = Klebsiella spp. samples

34
 100
90
80
Zone of Inhibition (mm)

70
60
50 Chloramphenicol
Ciprofloxacin
40
Gentamicin
30 Tetracycline
20
10
0
K1 K2 K3 K4 K5 K6 K7 K8 K9 K10
Samples

Figure 2: Comparison of Diameter Zone of inhibition (mm) of Klebsiella spp. (K1-10) using the

prepared antibiotic discs of Tetracycline, Gentamicin, Ciprofloxacin and Chloramphenicol.

35
Table 5: Zone of Inhibition (mm) of other Antibiotics against Escherichia coli.

Escherichia coli Antibiotics Zone of Inhibition

Samples

T G CI CH

E1 13 35 10 32

E2 12 12 10 33

E3 11 38 18 18

E4 22 30 18 27

E5 25 17 17 12

E6 35 23 11 23

E7 16 22 12 31

E8 14 23 18 21

E9 13 24 29 14

E10 13 16 30 12

Keys:
T= Tetracycline CI= Ciprofloxacin
G= Gentamicin CH= Chloramphenicol
E1-10= Escherichia coli samples

36
 100
90
80
Zone of Inhibition (mm)

70
60
50 Chloramphenicol
Ciprofloxacin
40
Gentamicin
30 Tetracycline
20
10
0
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10
Samples

Figure 3: Comparison of Diameter Zone of inhibition (mm) of Escherichia coli (E1-10) using the

prepared antibiotic discs of Tetracycline, Gentamicin, Ciprofloxacin and Chloramphenicol.

37
4.2 Interpretation for the CEFTRIAXONE Disc Zones of Inhibition

The result for the interpretation of the antibiotic activity of the prepared

ceftriaxone antibiotics against Klebsiella spp. and Escherichia coli samples

presented in Table 2 and 3 shows, Klebsiella samples (K1-10) were resistant to the

ceftriaxone antibiotics but sensitive to the other antibiotics, as shown in Table 4.

Similarly, Escherichia coli samples (E1-10) were resistant to some ceftriaxone

antibiotics but sensitive to the other antibiotics, as shown in Table 5.

According to the Clinical Laboratory Standard Institute (CLSI) standard, diameter

zone of inhibition ≤ 19mm indicates resistance, hence the 0mm measured on both

the Klebsiella and Escherichia coli samples (K1-10) and (E1-10), showed zero

clearance against the Ceftriaxone antibiotic.

38
Plate 2: Ceftriaxone against Escherichia coli (E1)

39
Plate 3: Ceftriaxone against Klebsiella spp. (K1)

40
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMENDATIONS

5.1 Discussion

The findings of the study on the effect of the prepared antibiotics Ciftriaxone on

Klebsiella spp. and Escherichia coli. in Table 3 and 4, revealed that the zone of

inhibition is 0mm. No clearance or activity to the meropenem antibiotic. This

suggests a high level of resistance within these strains to Ceftriaxone, which is

concerning as ESBLs are known for their ability to resist a wide range of

antibiotics.

In this study, the ESBL is most likely the mechanism at work. It is advisable to

propose that any of the mechanisms described by Datta et al. (2019), namely

overexpression of the efflux pump, β-lactamase production together with porin

loss, and ESBL, could be in order of causality.

Furthermore, the discovery of ESBL-resistant microbes at the University of Abuja

Teaching Hospital (UATH) supports Logan and Weinstein's (2017) assertion that

organisms capable of manufacturing ESBL can be found on every continent.

41
5.2 Conclusion and Recommendations

The detection of ESBL among Enterobacteriaceae is crucial in managing antibiotic

resistance and controlling the spread of multidrug-resistant bacteria.

The resistance profile of ESBL producing Enterobacteriaceae, shows that all

strains (K1-10 and E1-10) were 100% resistant to the Ceftriaxone antibiotic.

It is important to consider alternative treatment options and infection control

measures when dealing with such highly resistant strains to prevent the spread of

these bacteria and to effectively treat infections.

42
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Appendices
Appendix 1

48
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