Bacteriological analysis and antibiotic resistance in patients with suspected septicemia in Dhaka, Bangladesh (2025)

Declaration

It is hereby declared that

1. The thesis submitted titled “Bacteriological analysis and antibiotic resistance in patients

with suspected septicemia in Dhaka, Bangladesh” is our own original work while

completing a degree at Brac University.

2. The thesis does not contain material previously published or written by a third party, except

where this is appropriately cited through full and accurate referencing.

3. The thesis does not contain material which has been accepted, or submitted, for any other

degree or diploma at a university or other institution.

4. We have acknowledged all main sources of help.

Student’s Full Name & Signature:

Afsana Ahmed Sohana

Fahmida Ahmed

20226001

21326039

Mahanaz Mum Tarin Spriha

20326015

Approval

The thesis titled “Bacteriological analysis and antibiotic resistance in patients with

suspected septicemia in Dhaka, Bangladesh” submitted by

1. Afsana Ahmed Sohana (20226001)

2. Fahmida Ahmed (21326039)

3. Mahanaz Mum Tarin Spriha (20326015)

In Spring, 2025 has been accepted as satisfactory in partial fulfillment of the requirement for

the degree of Bachelor of Science in Microbiology in Fall, 2024

Examining Committee:

_______________________________

Supervisor:

Fahim Kabir Monjurul Haque, PhD

(Member)

Associate Professor of Microbiology Program &

Department of Mathematics and Natural Sciences

Brac University

_______________________________

Program Coordinator:

Nadia Sultana Deen, PhD

(Member)

Associate Professor of Microbiology Program &

Department of Mathematics and Natural Sciences

Brac University

Departmental Head:

_______________________________

(Chair)

Md. Firoze H. Haque, PhD

Associate Professor and Chairperson,

Department of Mathematics and Natural Sciences

Brac University

Ethics Statement

This study was conducted in strict accordance with the highest ethical standards in research. Blood

samples were collected from patients at four branches of Islamic Bank Hospital in Dhaka,

Bangladesh, with full adherence to established ethical guidelines. Ethical approval was sought and

granted from the review boards of all four hospital branches prior to the initiation of the study.

These institutional review boards meticulously reviewed and approved the research protocols to

ensure compliance with the National Guidelines on Health Research and the International

Guidelines for Ethical Considerations in Research.

Furthermore, the study underwent review and received approval from the Ethics and Research

Review Committee of the Faculty of Mathematics and Natural Sciences, MNS, at BRAC

University, Dhaka, Bangladesh. The research was conducted under the supervision of Dr. Fahim

Kabir Monjurul Haque, with all aspects of the study aligning with the ethical guidelines set forth

by BRAC University.

Informed consent was obtained from the patients prior to the collection of samples. patients were

thoroughly informed about the study's objectives, the procedures involved, potential risks, and the

benefits. Data confidentiality and patient’s anonymity were rigorously maintained, with all

personal identifiers being removed during data analysis.

This robust ethical framework underpins the study's integrity and validity, ensuring adherence to

internationally recognized norms and principles for ethical research.

Acknowledgement

We would like to begin by expressing our deepest gratitude to Almighty Allah for granting us the

opportunity to undertake and complete this research. Our heartfelt thanks go to our parents whose

unwavering support has been invaluable from the inception to the completion of this study.

We are also deeply grateful to the Chairperson of the Department of Mathematics and Natural

Sciences, Md. Firoze H. Haque, and to all the faculty members of BRAC University for their

continuous support. Special thanks to our supervisor, Dr. Fahim Kabir Monjurul Haque,

Associate Professor in the Microbiology Program, Department of Mathematics and Natural

Sciences, BRAC University. His expert guidance, constructive feedback, and unwavering support

were crucial to the success of this research. This work would not have reached its full potential

without his dedicated mentorship and insightful critiques.

Lastly, I want to show my heartfelt appreciation to my mentor, Fahim Faisal Nakib, for his

constant clear direction and guidance. His guidance helped me ease up and navigate the journey

while enlightening and enriching the experience.

Abstract

The objective of our study was to isolate bacteria from septicemia patient’s blood and subsequently

analyze their antibiotic resistance pattern. We collected 80 blood samples from four different

branches of Islami Bank Hospital (Mirpur, Kakrail, Motijheel, and Nayapaltan) in Dhaka,

Bangladesh. Isolating one bacterial colony from a blood culture plate of selective media. Then,

based on colony morphology, selecting the bacterial colonies and subculturing them for

presumptive identification. Confirming their identity through biochemical tests (catalase test,

oxidase test) and molecular test (polymerase chain reaction) followed by gel electrophoresis (for

visualizing the band size of targeted microorganisms). Among the 80 samples, 66 samples were

culture positive. From positive cultures, 65.2% were Salmonella typhi, 13.6% were Klebsiella

pneumoniae, 12.1% were Escherichia coli, and 9.1% were Staphylococcus aureus. 22% were

gram-positive and 77% were gram-negative) For antibiotic susceptibility tests (AST), we selected

one isolate from each culture-positive sample. Total 8 Escherichia coli, 43 Salmonella typhi, 9

Klebsiella pneumoniae, and 6 Staphylococcus aureus isolates were selected and AST was

performed using the Kirby-Bauer disc diffusion method. We observed that 90% to 100% of the

Salmonella typhi isolates 89% and 79% were resistant to Nalidixic acid and Ampicillin

respectively. 68%, 79% and 39 % of Klebsiella pneumoniae isolates were resistant to Amoxicillin,

Cefixime and Erythromycin respectively. 69% and 80% of all Escherichia coli isolates were

resistant to Amoxicillin and Cefixime respectively, and 28% of the isolates were resistant to

Azithromycin, Ciprofloxacin, Imipenem, Ceftriaxone. 49% of all Staphylococcus aureus isolates

were resistant to Cefixime, Sulfamethoxazole and Ceftazidime,

28% were resistant to

Erythromycin and Ceftriaxone, and 19% were resistant to Imipenem and Gentamicin. In this study,

a total of 18 isolates were identified as multi-drug resistant (MDR), including 7 Salmonella typhi,

6 Klebsiella pneumoniae, 2 Escherichia coli, and 3 Staphylococcus aureus strains. Among these,

3 isolates were further classified as extensively drug-resistant (XDR), comprising 1 Salmonella

typhi, 1 Klebsiella pneumoniae, and 1 Staphylococcus aureus isolate.

Keywords: Septicemia, Salmonella typhi, Klebsiella pneumoniae, Escherichia coli,

Staphylococcus aureus, Multi-drug resistant (MDR), Extensively drug-resistant (XDR).

List of Figures:

Figure No.

Figure Title

Page No.

Different bacterial colonies of different selective media

2.4

Different type of biochemical tests

2(a).

Agarose gel electrophoresis of PCR assay of Klebsiella

pneumoniae isolates

2(b).

Agarose gel electrophoresis of PCR assay of Staphylococcus

aureus isolates

2(c).

Agarose gel electrophoresis of PCR assay of Salmonella typhi

isolates

2(d).

Agarose gel electrophoresis of PCR assay of Escherichia coli

isolates

Prevalence of Isolated pathogens in Septicemia Patients based

on positive results

4(a).

Antibiotic Susceptibility Profiling of Salmonella typhi

positive isolates

4(b).

Antibiotic Susceptibility Profiling of Klebsiella pneumoniae

positive isolates

4(c).

Antibiotic Susceptibility Profiling of Escherichia coli

positive isolates

4(d).

Antibiotic Susceptibility Profiling of

Staphylococcus aureus positive isolates

CHAPTER 1

1.1: Introduction

Sepsis is a hazardous organ dysfunction caused by an immune response to infection. And so it is

a leading cause of death, especially in low income countries like Bangladesh. In developing

countries like Bangladesh, India or Pakistan, the leading causes of infection are malaria,

pneumonia and diarrhea. Here, sepsis dominates because of higher malnutrition, lack of certified

doctors, poor healthcare infrastructure, and late sepsis detection. If left untreated, sepsis can

manifest in more severe forms like septic shock, multiple organ failure, and finally death. That’s

why it is important to detect sepsis as soon as possible. We also need more information to design

the most appropriate treatment method for each sepsis case. (Garbern et al., 2024) At present,

special colorimetric assays can detect septicemia at early stages, by tracking special biomarkers in

blood. This is a very important discovery for medical science. Because a patient’s life depends on

the earliest possible availability to treatment. (Dashtian et al., 2024). This has been confirmed in a

review of 42 studies on 19000 patients and more. All the findings confirm that the death risks of

septicemia drop by 16 percent if the patients receive treatment within the first 3 hours of diagnosis.

After this time has passed, the chances of death increase by 30 per cent. The results are published

in the European Journal of International Medicine (Leung et al., 2024). But sadly, antibiotic

resistance is on the rise (confirmed by the World Health Organization). This is a huge obstacle to

treatment (Baral et al., 2024). And most of the causative agents of septicemic pathogens are already

resistant to the commonly used antibiotics. For example, a study in Turkey shows that deaths of

48% of infected patients are caused by gram negative sepsis. Infections from Methicillin resistant

Staphylococcus aureus cause 36 % of deaths. Carbapenem-resistant pathogens can kill 60 percent

of patients on average. (Gucer et al., 2024). Another research in Ethiopia showed that 89 percent

of nosocomial pathogens are already resistant to multiple drugs (like ampicillin or amoxicillin.)

Among them, 34.7% are resistant to carbapenems and 46.3 percent to beta lactams (Tilahun et al.,

2024). These statistics are concerning. So, these pathogens should be studied extensively. And

appropriate antibiotics should be designed to stop their growth, so that antibiotic resistance can be

slowed down or reversed, if possible. (Baral et al., 2024). Even Bangladesh is trying to design

proper treatment plans for septicemia. Experts from other countries are also trying to find solutions

in our country, like Dr. Stephanie Garbern from Brown University, United States. She mainly

studied children affected with septicemia in poor and middle-income countries. She even worked

with engineers and local doctors to develop an effective diagnostic gadget. It was also trained with

a machine learning algorithm, which can collect the person’s data (heart rate, oxygen rates etc.)

Then the AI can detect early symptoms of sepsis, which doctors might mistake as fever or common

cold. She only used cheap, available tools so that poor and middle-class people can access it too.

(Garbern et al., 2024). Nevertheless, it is important to find cost effective ways to diagnose and

treat septicemia in a third world country like Bangladesh.

Despite the discovery of advanced treatment methods for septicemia, antibiotics are the standard

treatment plan because it directly targets the pathogens, who are the main agents of disease. That’s

why we felt the need to conduct this research- to find the ideal antibiotic to kill each pathogen. But

antibiotics are losing their potency because of some malpractices in our Healthcare system.

Antibiotic usage is largely unregulated in Bangladesh, especially in rural areas. Without diagnosis,

pharmacists and doctors prescribe multiple antibiotics to the customers without diagnosis. These

antibiotic sellers don't know the guidelines of using antibiotics. In addition, patients don't complete

their medication for the full prescribed time, which buys time for bacteria to develop antibiotic

resistant genes (Bepari et al., 2023). Extensive studies have been conducted on this pattern of

emergence. A review article analyses 46 studies from 2014 to 2018, which reveals high prevalence

of antibiotic resistance among common pathogens. (Ahmed et al., 2019).

Regarding the growing resistance to antibiotics, it is essential to conduct regular studies to identify

the bacteria causing septicemia among Bangladeshi patients and assess their antibiotic resistance.

This helps to monitor the changes in antibiotic resistance. Additionally, this will provide doctors

and authorities with insights to develop treatment strategies that prescribe efficient antibiotics for

septicemia patients.

1.2: Objectives

The objectives of this study were to isolate the bacteria causing septicemia from septicemia-

suspected patients in Dhaka city and to analyze their antibiotic resistance patterns.

CHAPTER 2

Materials and Methods

2.1 Study design

The study was conducted among clinically suspected septicemia patients who were admitted to

hospitals in Dhaka, Bangladesh. Samples were collected from four branches of Islami Bank

Hospitals; Mirpur, Motijheel, Kakrail and Naya paltan & 80 samples were collected from these

branches. The blood sample collection duration was from December,2024 to April, 2025. Blood

samples were collected from patients of various age groups and both genders who were admitted

to various wards including the Intensive Care Unit (ICU), Medicine, Pediatrics, and Emergency

wards. Written consent was taken prior to sample collection, from either patients or the legal

guardians of patients.

2.2 Sample collection

Venous blood samples were taken from clinically confirmed septicemia patients upon admission

to hospitals that were chosen for the study. Venipuncture was used to obtain blood samples using

sterilized, disposable syringes and needles by professional experts. Before beginning antibiotic

treatment, each patient had a blood draw of about 2-3 mL. Blood was immediately put into a

heparin tube which was transported to the microbiology lab via a sealed iced box(4°C). The

samples were appropriately labeled with the information of patient's ID, date, name, gender and

indoor/outdoor patients. After being collected, they were taken to the microbiological lab in 1-2

hours(Nursing et al., 2023).

2.3 Culture to isolate the bacteria

Upon arrival at the laboratory, each blood sample was immediately processed to ensure the

integrity of microbial content. A volume of 100 µL of the blood sample was aseptically dispensed

and spread onto various culture media. This was carried out using a sterile micropipette and

spreader under laminar flow conditions to minimize contamination. The selected media types

included MacConkey (MAC), Xylose Lysine Deoxycholate (XLD), Eosin Methylene Blue

(EMB), Mannitol Salt Agar (MSA), HiCrome™ UTI, HiCrome™ KPC Agar media. They were

then incubated at 37°C and checked 5-7 days for indications of microbial growth such as changes

in medium color and colonies. After up to seven days of incubation, spread plates were observed

if there is any growth. Positive cultures that showed evidence of growth were sub cultured onto

selective and differential media such as Salmonella typhi on SS Agar media, XLD medium,

Escherichia coli on EMB agar, HiCrome™ UTI Agar, klebsiella pneumoniae on KPC,

Staphylococcus aureus on Mannitol Salt Agar. Thus, expected colony morphology of these

organisms has shown in Table 1. For 18 to 24 hours, plates were incubated at 37°C, and the visible

of bacterial colonies was observed and has shown in the Figure 1.

Table 1: Desired colony morphology of isolated bacteria

Organism

Gram

Media

Expected Colony

Positive/Negative

Morphology

Staphylococcus aureus

Gram-positive

Yellow/white colonies

Mannitol Salt Agar

surrounded by the yellow

zone

Klebsiella pneumoniae

Gram-negative

HiCrome UTI Agar

Blue, mucoid

Klebsiella pneumoniae

Gram-negative

HiCrome KPC Agar

Bluish-green

Escherichia coli

Gram-negative

HiCrome UTI Agar

Purple colonies

Escherichia coli

Gram-negative

EMB Agar

Purple with a black center

and green metallic sheen

Salmonella Shigella

Salmonella typhi

Gram-negative

Agar

Colorless with black centered

Xylose-Lysine-

Salmonella typhi

Gram-negative

Red with black centered

Deoxycholate agar

Figure 1 : Different bacterial colonies of different selective media. (A) Salmonella typhi has given

black centered on XLD agar, (B) Escherichia coli shows green metallic sheen on EMB agar, (C)

Staphylococcus aureus gives yellow colored colony on MSA, (D) Escherichia coli has given

purple colony on HiCrome UTI Agar, (E) Klebsiella pneumoniae has given blue colony on

HiCrome KPC Agar & lastly (F) Escherichia coli with purple colony & Klebsiella pneumoniae

with blue colony which distinguish them on UTI media.

2.4 Biochemical tests

Biochemical tests were performed to analyze the metabolic and enzymatic properties of the

isolated organisms. Here, catalase, oxidase tests were done for Staphylococcus aureus, Escherichia

coli, Salmonella typhi & Klebsiella pneumoniae. Table 2 shows the biochemical test results for

the isolated bacteria.

Catalase test method:

1)Spread the bacteria on an agar plate and incubate it overnight (18-24 hours) under the proper

conditions.

2)Using a sterile inoculating loop, select bacteria from a single colony and transfer them to a

microscope slide.

3)After adding one drop of 3% H2O2 to the bacteria, the suspension will give the result whether

it is positive or negative.

● Positive test result: Gas formation (O2) in the form of bubbles shows that the bacterium

has a catalase.

● Negative test result: No Gas formation (O2) in the form of bubbles.

Oxidase test method:

1)On a piece of filter paper, apply two drops of the oxidase reagent.

2)Using a sterile loop to transfer bacteria from one colony onto the oxidase reagent area. The result

can be observed after 30 sec.

● Positive test result: Dark blue-purple color change within 10-30 sec.

● Negative test result: No color change or color change after more than 30 sec (VETBact,

n.d).

2.5 DNA extraction

DNA was extracted for the purpose of identifying the organism by visualizing their band

production in gel electrophoresis followed by PCR. For the isolation of high-quality bacterial

genomic DNA, a freshly grown subculture was prepared using nutrient-rich growth media. As a

base solution for cell lysis, 1X Tris-EDTA buffer was prepared before the DNA extraction process

started. First, an Eppendorf tube was filled with 400 µL of 1X TE buffer. The buffer-containing

tube was then inoculated with a loopful of the bacterial culture. After that, the sample was then

vortexed to thoroughly mix the TE buffer with the bacterial cells for 15 seconds. To pellet the

bacterial cells, the sample was centrifuged for 10 minutes at 13,000 rpm and 25°C after initial

mixing. After centrifugation, the supernatant was discarded and 400 µL of 1X TE buffer was added

to the Eppendorf tube along with the pellet. The bacterial pellet was then resuspended by vortexing

the sample once more. In order to rupture the bacterial cells and liberate their genomic DNA, the

samples were to undergo a heat treatment. For ten minutes, the tubes were kept at 100°C in a heat

block machine. The bacterial cell wall and membrane will be properly lysed by this heat treatment,

releasing the DNA into the solution. Following this heat block process, the samples were allowed

to cool for five minutes at room temperature. To remove the recovered DNA from any leftover

debris or biological components, the cooled samples were centrifuged for 10 minutes at 13,000

rpm. Lastly, 200 µL of the DNA supernatant was transferred to a new Eppendorf tube after these

centrifugation procedures. Extreme caution was used to avoid disturbing the pellet at the tube's

bottom during the transfer. The isolated DNA was now present in this supernatant, making it

suitable for additional examination. Then store the DNA at -20° temperature. For use in PCR or

sequencing, this technique guaranteed the effective extraction of high-quality DNA from the

bacterial cells (Moore et al,2005).

2.6 PCR (Polymerase Chain Reaction)

To confirm the identity of the bacterial isolates, Polymerase Chain Reaction (PCR) was performed.

This molecular method was utilized to detect organism-specific genetic sequences.

PCR method

PCR cycle is carried out 25-40 times in order to amplify the DNA according to the organisms. To

initiate the PCR process, each reaction mixture was prepared in a final volume of 13 μL. The

reaction mixture included 3 μL of nuclease-free water, 1 μL of forward primer, 1 μL of reverse

primer, 6 μL of Emerald Amp PCR Master Mix (Takara Bio Inc.), and 2 μL of template DNA. All

components were carefully added into sterile PCR tubes. Following preparation, the tubes were

vortexed gently for 3-5 seconds to ensure thorough mixing of reagents. The mixtures were then

briefly centrifuged to collect contents at the bottom of the tubes before being loaded into a thermal

cycler (Applied Biosystems 2720 Thermal Cycler). PCR amplification was carried out under

standardized cycling conditions. Typically, 25-35 amplification cycles were performed which

involved denaturation, primer annealing, and extension steps-allowing exponential amplification

of the target DNA region. The total run time for the PCR ranged from 1.5 to 3 hours, depending

on the specific protocol and target DNA fragment length. Upon completion, all amplified PCR

products were stored at -20°C to preserve sample integrity for downstream applications such as

gel electrophoresis or sequencing (Lorenz, 2012).

Targeted genes and their primers:

This study focused on detecting virulence-associated genes specific to Escherichia coli, Klebsiella

pneumoniae, Staphylococcus aureus, and Salmonella Typhi in order to screen blood samples for

the presence of pathogenic strains, making it essential to identify the genetic markers associated

with their pathogenicity. For example, these four microorganisms are prominent etiological agents

in septicemia, each with distinct virulence factors. Table 3 shows the list of targeted genes &

primers of these organisms. This investigation aimed to detect these bacterial pathogens in blood

samples by targeting their specific virulence genes, thereby contributing to a better understanding

of the microbial causes of septicemia.

Table 3: List of targeted genes and of their primers

Name of

Primer

Targeted genes

Primer Sequence

Prod

PCR conditions

References

bacteria

Designati

(5′to 3′)

uct

size

Klebsiella

KP pf-F

16S-23S rRNA

ATTTGAAGAGGT

130

The cycling conditions were 10

(Kot et al.,

pneumoni

Internal

TGCAAACGAT

min at 94°C followed by

2023)

Transcribed

cycles of 30 s at 94°C, 20 s at

Spacer (ITS)

57°C and 20 s at 72°C, followed

by a 10 min final extension 72°C.

KP pr1-R

16S-23S rRNA

TTCACTCTGAAG

Internal

TTTTCTTGTGTTC

Transcribed

Spacer (ITS)

Escherich

ECO- F

malB (maltose

GACCTCGGTTTA

585

Initial denaturation at 95°C for 5

(Lindsey et

ia coli

operon)

GTTCACAGA

min; 35 cycles of denaturation at

al., 2017)

94°C for 45s, annealing at 45°C

for 45s, and extension for 1 min

followed by a final extension at

ECO-R

malB (maltose

CACACGCTGACG

72°C for 5 min.

operon)

CTGACCA

Salmonell

invA

GTATTGTTGATT

403

Initial denaturation at 95°C for 5

(Ranjbar et

a typhi

a-F

AATGACATCCG

min; 35 cycles of denaturation at

al., 2016)

95°C for 30s, annealing at 60°C

for 30s, and extension for 1 min

72°C followed by a final

Salmonell

invA

ATATTACGCTAC

extension at 72°C for 10 min.

a-R

GGAAACACGTT

Staphyloc

NUC-F

nuc

GCGAT

279

4 min at 94°C and then 34 cycles

(Gogoi et

occus

GATGGTGATACG

of 1min at

94°C for

the

al., 2024)

aureus

GTT

denaturation step and

45s at

55°C for the annealing &

extension at

72°C for

45s

followed by final extension at

NUC-R

nuc

AGCCAAGCCTTG

72°C For 10min.

ACGAACTAAAGC

2.7 Agarose gel electrophoresis

After performing Polymerase Chain Reaction, the resulting DNA fragments were analyzed

through Agarose gel electrophoresis method. This technique was employed to visualize DNA

fragment sizes and confirm the presence of DNA by comparison with a DNA ladder. To prepare

the gel, agarose powder (typically 1.5-2%) is mixed with 2000 μL of 50x Tris-acetate-EDTA

(TAE) buffer and distilled water (98%) and heated until fully dissolved. Ethidium bromide, an

intercalating agent that fluoresces under UV light, is added at a concentration of 4 μL to enable

DNA visualization. PCR products are carefully loaded into the wells of the prepared gel using

sterilized (autoclaved) micropipette tips. The gel is run using a 10x TAE buffer as the running

buffer, facilitating the movement of DNA toward the positive pole. For each run, 4 μL of a 100 bp

DNA ladder (Biolab) is used as a reference and electrophoresis is carried out at 100 volts at 400

amp. Once the electrophoresis is complete, the gel is observed under UV light. The DNA bands

become visible due to binding of the fluorescent stain, which emits specific colors. Band sizes are

then determined by comparing them to the bands in the DNA ladder, and the results are recorded

accordingly (Addgene: Protocol - How to Run an Agarose Gel, n.d.). Below the four types of band

sizes have been shown in Figure 2(a), 2(b), 2(c), and 2(d).

Figure 2(a): Agarose gel electrophoresis of PCR assay of Klebsiella pneumoniae isolates. Here,

first lane is 100 bp DNA ladder, 2nd lane is negative control and well 3-14 are positive samples at

130 bp. The image was captured using a camera while the agarose gel was positioned under a UV

transilluminator. The complete original image was utilized to generate the figure panel.

Figure 2(b): Agarose gel electrophoresis of PCR assay of Staphylococcus aureus isolates. Here,

first lane is loaded with a 100 bp DNA ladder, the 2nd lane is negative control and lanes 3-11 are

positive samples at 279 bp. The image was captured using a camera while the agarose gel was

positioned under a UV transilluminator. The complete original image was utilized to generate the

figure panel.

Figure 2(c): Agarose gel electrophoresis of PCR assay of Salmonella typhi isolates. Here, first

lane is 100 bp DNA ladder, 2nd lane is negative control and lane no. 3-10 are positive samples at

403bp. The image was captured using a camera while the agarose gel was positioned under a UV

transilluminator. The complete original image was utilized to generate the figure panel.

Figure 2(d): Agarose gel electrophoresis of PCR assay of Escherichia coli isolates. Here, first

lane is a 100 bp DNA ladder, the second lane is negative control and well 3-12 are positive samples

at 585 bp. The image was captured using a camera while the agarose gel was positioned under a

UV transilluminator. The complete original image was utilized to generate the figure panel.

2.8 Antibiotic susceptibility testing

The Antibiotic Susceptibility Test (AST) was performed using the Kirby-Bauer disk diffusion

method to determine the effectiveness of various antibiotics against the isolated bacteria. Each

bacterial isolate from the culture plates was sub-cultured on freshly prepared nutrient agar to obtain

a fresh and pure culture before performing AST. Mueller-Hinton Agar (MHA) media was freshly

prepared for antibiotic susceptibility testing. A 0.9% sodium chloride (NaCl) solution was also

prepared by dissolving the appropriate amount of NaCl in distilled water and briefly heating it.

Using a sterile glass pipette, 5-6 mL of the NaCl solution was dispensed into each test tube. A

bacterial suspension was prepared by picking colonies from a pure culture and adjusting the

turbidity to match the 0.5 McFarland standard. The suspension was then evenly spread over the

surface of a Mueller-Hinton agar plate using a sterile cotton swab to ensure a uniform lawn of

bacterial growth. After allowing the plate to dry for a few minutes, antibiotic discs were placed on

the agar surface using sterile forceps. The plates were incubated at 35-37°C for 16-18 hours (Jan

Hudzicki, 2009).

Zone Of inhibition:

Following 24 hours of incubation, the Mueller-Hinton Agar (MHA) plates were examined for

bacterial growth surrounding the antibiotic discs. If an antibiotic effectively inhibits the test

organism, a clear area devoid of bacterial growth-known as the zone of inhibition-will be

observed around the disc. In contrast, if the antibiotic is less effective or ineffective, bacterial

growth will extend closer to the disc, resulting in a smaller or no visible inhibition zone. The

diameter of each zone of inhibition is measured using a ruler or scale to accurately assess the

degree of bacterial growth suppression. These measurements are then interpreted using

standardized reference charts provided by Oxoid Limited (England), which classify the bacteria as

sensitive, intermediate, or resistant to the antibiotics tested. This interpretation method aligns with

the guidelines recommended by the National Antimicrobial Resistance Monitoring System and the

Clinical and Laboratory Standards Institute (CLSI, 1997, 1999). Table 4: shows the concentration

and interpretive criteria for diffusion zones of the antibiotics.

Table 4: Concentrations and interpretive criteria for diffusion zones of the antibiotics.

Antibiotic

Group

Effective against

Disc

Interpretative Criteria

code

potency

Sensitive

Intermediate

Resistant

(µg)

mm or more

mm or less

Azithromycin

Macrolide

Gram-positive

and

AZM

14-17

Gram-negative

Amikacin

Aminoglycoside

Gram-positive

and

15-16

Gram-negative

Colistin

Polymyxin E

Gram-negative

11-17

Cefepime

Cephalosporin

Gram-positive

and

CPM

19-24

Gram-negative

Meropenem

Carbapenem

Gram-positive

and

MEM

20-22

Gram-negative

Imipenem

Carbapenem

Gram-positive

and

IMI

20-22

Gram-negative

Nalidixic Acid

Quinolone

Gram-negative

14-18

Streptomycin

Aminoglycoside

Gram-positive

and

12-14

Gram-negative

Ampicillin

Beta-lactamase

Gram-positive

and

AMP

14-16

Gram-negative

Amoxicillin

Beta-lactam

Gram-positive

and

AML

10 µg

14-16

(Penicillin)

Gram-negative

Vancomycin

Glycopeptide

Gram-positive

15-16

Linezolid

Oxazolidinones

Gram-positive

21-22

Tigecycline

Glycylcycline

Gram-positive

and

TGC

15-17

Gram-negative

Tetracycline

Tetracycline class

Broad-spectrum (Gram-

12-14

positive and Gram-

negative)

Ciprofloxacin

Fluoroquinolone

Broad-spectrum Gram-

CIP

16-20

negative

Chloramphenicol

Amphenicol

Broad-spectrum (Gram-

13-17

positive and Gram-

negative)

Cefixime

3rd

Gen

Gram-negative

CFM

16-18

Cephalosporin

Ceftazidime

3rd

Gen

Gram-negative

CAZ

15-17

Cephalosporin

Erythromycin

Macrolide

Mainly Gram-positive,

14-22

some Gram-negative

cocci

Gentamicin

Aminoglycoside

Broad-spectrum;

13-14

effective mainly against

Gram-negative

and

some

Gram-positive

bacteria

Trimethoprim-

Sulfonamide

Broad-spectrum; many

SXT

11-15

Sulfamethoxazole

combination

Gram-positive

and

Gram-negative bacteria

CHAPTER 3

Result

3.1 Demographic characteristics of patients

This study included a total of 80 participants, individuals sourced from Islami Bank Hospital from

Mirpur, Motijheel, Kakrail, Naya Paltan branches of Dhaka. Among these participants,

15(18.75%) from Islamic Bank Hospital Motijheel branch, while 40 (50%) were from Mirpur

branch & the remaining 15(18.75%) & 10(12.5%) patients from Naya Paltan branch & Kakrail

branch. The majority of the study’s subjects fell within the age range of 40 to 80 years old.

Moreover, male participants take more account for this study with a percentage of 56.25% &

remaining 43.75% is female participants.Among the patients, most of them acquired infection

through community & after that they got admitted to the hospitals. A summary of the demographic

characteristics of the study’s patients is presented in Table 5.

Table 5: Demographic characteristics of patients

Characteristics

Categories

Frequency

Percentage

Number of participants

Islamic Bank Hospital, Mirpur

50%

at this study

Islamic Bank Hospital, Motijheel

18.75%

Islamic Bank Hospital, Kakrail

12.5%

Islamic Bank Hospital, Nayapaltan

18.75%

Gender

Male

56.25%

Female

43.75%

Age(in years)

0-19

12.5%

20-39

18.75%

40-59

25%

60+

43.75%

Patient type

In patient

31.25%

Outpatient

68.75%

3.2 Prevalence of positive isolates in septicemia patients

Among 80 samples, bacterial growth was observed in 66 samples. The distribution of the bacteria

in these culture-positive samples are shown in Figure 3. The data indicates that Salmonella typhi

is the most prevalent pathogen, responsible for 65.2% of the positive isolates, with 43 out of 80

samples yielding positive results. Klebsiella pneumoniae follows with 9 positive isolates,

representing 13.6% of the total, while E. coli is identified in 8 isolates, accounting for 12.1%.

Staphylococcus aureus appears in 6 isolates, making up 9.1% of the cases. All infections were due

to a single organism. These findings reflect the distribution of bacterial pathogens in septicemia

cases and emphasize the dominance of Salmonella typhi in the observed patient population.

Figure 3: Prevalence of isolated bacteria in 66 culture-positive samples of suspected septicemia

patients. This pie chart shows the distribution of isolated pathogens identified in septicemia

patients. Salmonella typhi at 65.2% of the total isolates, followed by Klebsiella pneumoniae at

13.6%, Escherichia coli at 12.1%, and Staphylococcus aureus at 9.1%.

3.3 Antibiotic resistance & sensitive profile of the positive isolated organisms

In this study, a total of 80 isolates have been selected to identify the antibiotic resistance of

different organisms. The Kirby-Bauer Disc Diffusion test was performed on each isolate. A total

of 8 isolates of Escherichia coli, 43 isolates of Salmonella typhi, 9 isolates of Klebsiella

pneumoniae, and 6 isolates of Staphylococcus aureus were examined for antibiotic susceptibility.

Figure 4(a), 4(b), 4(c), 4(d) demonstrates all antibiotic susceptibility profiles for specific bacteria.

Figure 5 shows the MDR, XDR & susceptible of all the isolates. In order to get the best possible

treatment results, these findings highlight the significance of concentrating on highly efficient

antibiotics while avoiding those with high resistance.

Antibiotic Susceptibility Analysis of Salmonella typhi:

Out of the all-positive Salmonella typhi isolates showed relatively high antibiotic susceptibility,

with the exception of Nalidixic acid (89%) & Ampicillin (79%), which demonstrated substantial

resistance. The efficiency of these 12 antibiotics against the isolates is displayed in the graph

Figure 4(a) below. From the graph, it is evident that Chloramphenicol, Ceftazidime, Gentamicin,

Ceftriaxone and Meropenem showed high susceptibility, with 90% to 100% of the isolates being

susceptible.

Figure 4(a): Antibiotic Susceptibility Profiling of Salmonella typhi. Total isolate number was 43.

Antibiotic Susceptibility Analysis of Klebsiella Pneumoniae:

Most of the positive Klebsiella pneumoniae isolates showed in the relatively high antibiotic

susceptibility, with the exception of Amoxicillin (68%), Cefixime (79%) which demonstrated

substantial resistance & Erythromycin (39%) comparatively lower resistance. The efficiency of

these 12 antibiotics against the isolates is displayed in Figure 4(b) below. From the graph, it is

evident that Ciprofloxacin, Azithromycin, Gentamicin, Tetracycline, Tigecycline and Meropenem

showed high susceptibility, with 75% to 98% of the isolates being susceptible.

Figure 4(b): Antibiotic Susceptibility Klebsiella pneumoniae. The total number of isolates was 9

Antibiotic Susceptibility Analysis of Escherichia coli:

Out of the all-positive Escherichia coli isolates showed relatively high antibiotic susceptibility,

with the exception of Amoxicillin (80%) and Ampicillin (99%), which demonstrated substantial

resistance. Cefixime (58%), Ceftriaxone (28%) and Tetracycline (38%) resistance of positive

isolates. The efficiency of these 12 antibiotics against the isolates is displayed in the graph Figure

4(c) below. From the graph it is evident that Azithromycin, Chloramphenicol, Ciprofloxacin,

Imipenem, Colistin, Tigecycline, Gentamicin showed high susceptibility, with 100% of the isolates

being susceptible.

Figure 4(c): Antibiotic Susceptibility Profiling of Escherichia coli. The total number of isolates

was 8.

Antibiotic Susceptibility Analysis of Staphylococcus aureus:

Out of all positive Staphylococcus aureus isolates showed relatively high antibiotic susceptibility,

with the exception of Cefixime, Sulfamethoxazole and Ceftazidime showed 49% of resistance.

Then Erythromycin and Ceftriaxone showed 28% resistance among positive isolates. Imipenem

and Gentamicin showed 19% of resistance which is comparatively lower. The efficiency of these

12 antibiotics against the isolates is displayed in the graph Figure 4(d) below. From the graph it

is evident that Azithromycin, Ciprofloxacin, Tigecycline, Vancomycin, Tetracycline and

Imipenem showed high susceptibility, with 85% to 100% of the Positive isolates

Figure 4(d): The Graph showing the resistance and sensitivity to various antibiotics of

Staphylococcus aureus. The total number of isolates was 6

3.4: Multi-drug resistant bacteria

Among the all-positive isolates of Salmonella typhi shows 16.67% for MDR & 2.32% for XDR.

MDR in klebsiella pneumoniae isolates showed 66.67% with 11% XDR. Escherichia coli has

shown MDR in 25% but no XDR. Lastly, gram positive bacteria Staphylococcus aureus showed

MDR in 50% & 16.67% XDR. The graph (Figure 5) shown MDR and XDR pattern below,

Organism

MDR

XDR

Salmonella typhi(N=43)

7 (16.67%)

1(2.32%)

Escherichia coli(N=8)

2 (25%)

Klebsiella

6 (66.67%)

1(11%)

pneumoniae(N=9)

Staphylococcus

3 (50%)

1 (16.67%)

aureus(N=6)

Figure 5: The table showing MDR and XDR pattern

CHAPTER 4

Discussion

This study aimed to assess the bacteriological profile and antibiotic resistance patterns of

septicemia pathogens among diagnosed patients at the four branches of Islamic Bank Hospital in

Dhaka city, Bangladesh. The study's findings revealed that among a total of 80 samples,

Salmonella typhi was the most prevalent pathogen, accounting for 65.2% of the isolates, followed

by Klebsiella pneumoniae (13.6%), Escherichia coli (12.1%), and Staphylococcus aureus (9.1%).

This study aligns with the international and local patterns of Salmonella typhi, Klebsiella

pneumoniae, Escherichia coli, and Staphylococcus aureus being the predominant pathogens in

bloodstream infections (BSIs). The prevalence of Salmonella typhi in this study (65.2%) is

consistent with findings from Kathmandu, Nepal, where Salmonella typhi accounted for 71% of

blood culture isolates (Simkhada, 2016). The high prevalence of Salmonella typhi in both studies

could be attributed to the common occurrence of typhoid fever in countries with poor sanitation

and water contamination issues. Typhoid fever, which has not yet been eradicated, continues to

cause septicemia (Simkhada, 2016); therefore, it is not surprising that in this study Salmonella

typhi was the leading pathogen with 65.2% of the isolates. Dagnew et al. (2013) reported similar

results in Ethiopia and Nepal, where Salmonella typhi dominated the septicemia pathogen.

The prevalence of klebsiella pneumoniae in our study (13.6%) is much lower compared to the

reported for Sub-Saharan Africa (54.4%). Escherichia Coli in your study (12.1%) is slightly lower

than the study reported in Sub-Saharan Africa (18.4%) (Lester et al., 2019). According to our

analysis, the prevalence of Staphylococcus aureus was strikingly low at 9.1%, unlike other studies

conducted in Bangladesh which noted a higher prevalence where Staphylococcus aureus was more

often associated with septicemia. More specifically, this is one of the comparisons that can be

made regarding the use of bacterial infections in the current study and Staphylococcus aureus for

past investigations conducted in the same part of the world. In contrast to the studies conducted

in Mymensingh, Bangladesh, which reported a prevalence of approximately 20%, the current study

showed a lower prevalence of 9.1%. This is likely due to differences in hospital-based settings or

the specific antibiotic resistance trends governing the prevalence of Staphylococcus aureus

infections in the community. Various reasons explain this, including differences in the patient

populations, healthcare settings, or methodologies employed in the studies (Khan et al., 2024).

The study analysis showed that septicemia was primarily in the elderly (60 years and above) which

contributed to 43.75% of the cases followed by the 40-59 age group with 25%. This aligns with

other global reviewed article where in older adults (≥65) constitute 58-65% of sepsis cases (Starr

& Saito, 2014). Due to septicemia being more common amongst older adults because of the

immunosenescence- a weakening immune system due to aging-associated with heightened

vulnerability to infections (Opal et al., 2005). Therefore, the geriatric population continues to be

at risk to certain community infections notably caused by Salmonella typhi, Escherichia coli, and

Klebsiella pneumoniae which could lead to potentially devastating complications if they are not

treated promptly. On the basis of gender, the males in this study outnumbered the females, 56.25%

and 43.75% respectively. Male predominance in septicemia cases is consistently reported globally,

In Wuhan, study found 61% male, 39% female among 2,111 sepsis patients (Zhao et al., 2025).

This disparity also aligns with other studies which demonstrate that men are at higher risk of

septicemia because of the differences in immune responses (Pradipta et al., 2013). It is known that

women’s immune response is enhanced due to the presence of estrogens, thus giving women better

defenses against infections, particularly in community settings.

This study highlights the increasing trend of antimicrobial resistance in Bangladesh. Antimicrobial

resistance (AMR) remains a critical global health challenge, with developing countries like

Bangladesh facing particularly high rates due to factors such as unregulated antibiotic use, and

inadequate healthcare infrastructure. In this study, Salmonella typhi (65.2%), Klebsiella

pneumoniae (13.6%), and Escherichia coli (12.1%) were the most prevalent pathogens in

septicemia patients, reflecting similar patterns observed in other countries. For instance, in Nepal,

Salmonella typhi was responsible for 71% of bloodstream infections (Simkhada, 2016), while in

Ethiopia, Klebsiella pneumoniae and Escherichia coli were also leading causes of sepsis, with

resistance rates of 68% to amoxicillin and 79% to cefixime (Dagnew et al., 2013). In our studies,

Salmonella typhi showed resistance to nalidixic acid (89%) and ampicillin (79%). Resistance to

commonly used antibiotics like ampicillin, azithromycin, and nalidixic acid in this study mirrors

trends in India, where Klebsiella pneumoniae and Escherichia coli show similar resistance patterns

to beta-lactams and fluoroquinolones (Khan et al., 2022). However, the study found relatively low

prevalence of Staphylococcus aureus (9.1%), which contrasts with other studies in Bangladesh

and globally, where Staphylococcus aureus typically accounts for a larger portion of septicemia

cases (Khan et al., 2024). Additionally, colistin resistance observed in the study aligns with rising

concerns in countries like China and India, where colistin resistance is becoming an emerging

public health threat, especially in multidrug-resistant Gram-negative infections (Zhu et al., 2021).

In contrast, in this study colistin is a more effective antibiotic against Salmonella typhi, Klebsiella

pneumoniae, Escherichia coli, and Staphylococcus aureus. While resistance rates in high-income

countries like the U.S. are comparatively lower, the increasing global spread of MDR and XDR

pathogens highlights the urgency of implementing robust infection control, better surveillance

systems, and effective antibiotic stewardship programs worldwide (Smith et al., 2023). Multidrug-

resistant (MDR) pathogens are a growing global concern, as demonstrated by the high prevalence

of MDR Salmonella typhi, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus

in this study. Isolates of 16.67% Salmonella typhi were identified as MDR, with 2.32% classified

as extensive drug-resistant (XDR). Additionally, Klebsiella pneumoniae isolates were shown

66.67% of MDR, with 11% were XDR, while Escherichia coli isolates were shown 25% of MDR,

and Staphylococcus aureus isolates were shown 50% of MDR, with 16.67% of XDR. Similar

findings have been reported in other countries, such as India, where 50% of Salmonella typhi

isolates were MDR (Khan et al., 2022), and Ethiopia, where 40% of Klebsiella pneumoniae

isolates were resistant to multiple drugs (Dagnew et al., 2013). In contrast, the prevalence of MDR

strains in developed countries like the U.S. is relatively lower, with Klebsiella pneumoniae MDR

rates reported around 15-20% (Smith et al., 2023). MDR rate reported in a study from south India,

where 24.6% of Escherichia coli isolates from bloodstream infections showed multidrug resistance

(Babu et al., 2018). The global rise in MDR and extensive drug-resistant (XDR) pathogens

highlights the need for improved infection control, antibiotic stewardship, and timely diagnostics

to manage AMR effectively in both high-income and low-income countries (Bayot & Bragg,

2024).

CHAPTER 5

Conclusion

Our studies have shown that most of the pathogens are already resistant to antibiotics like

Amikacin, Gentamicin, Imipenem, Tigecycline, Linezolid and Vancomycin. Age, gender and

immunity might also play a role in the frequency of the onset of sepsis and the effectiveness of

antibiotics. In our study, most of the sepsis patients are more than 60 years old (43%) and

male(56.25%). The thickness of the peptidoglycan layer on the surface of bacteria may have an

impact on their pathogenicity. Our study showed that 77% of the patients were infected by gram

negative bacteria, especially Salmonella typhi. Nevertheless, data on antibiotic susceptibility is not

universal and permanent, because antibiotic susceptibility or resistance varies from strain to strain,

and the results will differ with variations in location or continent. In addition, the antibiotics that

are currently effective on a pathogen, may not retain the same efficacy after a few years. Some of

them may even gain multidrug resistance. This happens because antibiotics pose a selective

pressure on microbes, or which they have to survive by evolving with newer antibiotic resistance

genes, which spread rapidly within the microbial kingdom (through horizontal and vertical gene

transfer). Nevertheless, we need to continue a thorough longitudinal study with larger sample sizes

(annually if possible), to validate the findings of the research and get more insights.

CHAPTER 6

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