DIVISION OF PASIG CITY

BUTING SENIOR HIGH SCHOOL

A. Santos St., Buting, Pasig City
NATURAL SCIENCES DEPARTMENT
Academic Year 2022 - 2023

Antibacterial effectiveness of Calabash (Crescentia cujete)

against Salmonella typhimurium

Researchers:

Guzman, Justine Mark M.

Mendoza, Ma. Kathryn Ysabelle S.

Mitmug, Sohailah A.

Palanca, Daryll Jean B.

Regulto, Danicka Venice L.

Santoalla, Christian Jess D.

12 - STEM D
Inquiry, Investigation, and Immersion
 CHAPTER 1:

THE PROBLEM AND ITS BACKGROUND

This chapter includes the introduction, conceptual framework, statement of the

problem, scope and limitations, significance of the study, experimental design, and the

definition of terms used.

Introduction

Over the last few years, the maximum number of salmonella illnesses has substantially

increased. Salmonella has a low infection rate but because of how widely it is distributed over

the world, it has become a problem for the food industry. In the study of Kurtz et al. (2017),

salmonella infection is a major source of morbidity and mortality globally. The host immune

response differs depending on whether the infection is acute, localized, systemic, and

persistent. The immune response as well as the course of the bacteria are typically influenced

by the anatomical site of the infection. Salmonella bacteria have developed many strategies to

get around or manipulate immunity for their own benefit. Salmonella infections in people are

primarily brought on via food. Additionally, the food item itself affects how much Salmonella

will infect you. Salmonellae can infiltrate the food chain at any point, and depending on how

the food is processed, consuming a contaminated final product can have negative effects on

humans.

Apart from this, according to the Division of Infectious Disease, a serious threat to

children’s health is posed by salmonella infections everywhere. Typhoid illness, often known

as enteric fever, affects 17 million people and results in 600,000 fatalities each year. In addition

to causing 1.4 million infections annually in the US, nontyphoidal Salmonella is thought to be

the cause of 400 to 600 fatalities. Consuming food or beverages that have been infected with
feces is a common way to spread infection. Restaurants and fast-food outlets were the primary

sources of nontyphoidal Salmonella foodborne outbreaks. These outbreaks were frequently

linked to poor food handling, inadequate food storage, raw ingredients that had come into touch

with contaminated people, animals, or the environment, finished items, and foods. However,

as a result of consumer awareness of the hazards connected with chemical preservatives, there

has recently been an increase in public demand for medicinal plants. In most nations, food-

borne illness and nutritional losses are mostly caused by salmonella contamination of food

products. Furthermore, given the pathogenic microorganisms’ increasing drug resistance, it is

useful and possible to use plant-based natural antibacterial compounds as an alternative to

antibiotics (Kazeminia et al., 2017).

Moreover, the antibacterial compounds of plants have occasionally been derived and

placed as an effective medication in certain illnesses. Even recently, several important

allopathic drugs were produced from plants. For instance, secondary metabolites, which can

have a variety of pharmacological effects, are produced by all plants. Crescentia cujete is

known to contain a variety of bioactive compounds. These plants grow in a tropical dry

grassland habitat. The review emphasized that the majority of studies on Crescentia cujete have

come from Asia and South America, primarily the Philippines, Bangladesh, Peru, Brazil, and

India, with only one study coming from Africa (Journal of Medicinal Plants Studies, 2019).

Additionally, the antibacterial activity of Crescentia cujete was assessed using the disc

diffusion method in the study of Parvin, M. S., et al. (2015), using the cultures of Escherichia

coli and staphylococcus aureus. The results of this study showed that Crescentia cujete’s leaves

and bark had anti-inflammatory and antibacterial capabilities, indicating that they may be used

therapeutically to treat bacterial infections and can influence disease processes by causing

cellular membranes to become unstable.

 Thus, the researchers of this study aim to test the antibacterial ability of the calabash

(Crescentia cujete) fruit extract and its effectiveness against Salmonella typhimurium.

Conceptual Framework

The paradigm of the study in Figure 1 shows the key variable that will be investigated

in order to determine the effectiveness of the Crescentia cujete fruit extract in combating

Salmonella typhimurium.

Apply to Crescentia
cujete fruit extract
Control
Treatment 1 (50% fruit Crescentia cujete
extract) Kirby-Bauer Disk
Antibacterial
Diffusion Method Effectiveness Data
Treatment 2 (75% fruit
extract)
Treatment 3 (100% fruit
extract)

Figure 1. Conceptual Framework

In Figure 1, the Crescentia cujete fruit extract, as the independent variable, will be

investigated to examine its antibacterial effectiveness to treat Salmonella typhimurium. In the

input of the study, the researchers will use three different concentrations of the fruit extract and

an antibiotic, in which case the researchers will use Azithromycin as the control of the

experiment.
 In the process, the Kirby-Bauer disk diffusion method, also known as the disc diffusion

test, is intended to be used as the test to determine the resistance or sensitivity of the bacteria

to calabash fruit extract. Kirby-Bauer testing involves placing bacteria on a plate of solid

growth medium and adding filter paper disks that have been moistened with various

concentrations of fruit extract and the antibiotic azithromycin. Proceed with the incubation

period of 24 hours until the bacteria colonies grow inside of the petri dish. In the end, the results

will be scrutinized by examining the cultural characteristics and zone of inhibition existing in

a petri dish.

The data acquired from the experiment will be the study's final product. The outcome

will compare the effects of azithromycin and the fruit extract of Crescentia cujete on

Salmonella typhimurium and validate the efficacy of Crescentia cujete in treating Salmonella

typhimurium at different concentrations of the fruit extract.

To evaluate if the bacterium is likely to be prevented from multiplying by an antibiotic,

a susceptibility test is employed. This test is carried out to examine whether different antibiotics

are effective against the bacteria and to see if they have become resistant to those particular

treatments. The test's outcome can be used to identify the treatment or antibiotic that will likely

be most successful in eradicating the bacteria (Testing.com, 2020).

The Susceptibility Test serves as the basis for this research as it discusses the reasoning

behind using the disk diffusion method to determine whether the antibiotic Azithromycin and

the fruit extract from the Crescentia cujete tree, which will be used to halt Salmonella

typhimurium from growing and multiplying. The Kirby-Bauer disk diffusion method has been

a standard for antibiotic susceptibility. It was initially created in the 1950s and improved by

W. A. Kirby. In 1961, the World Health Organization standardized it (Libretexts, 2021).

 The leaves and bark of Crescentia cujete were discovered to have antibacterial and anti-

inflammatory properties in the study conducted by Parvin et al. (2015), suggesting that the

plant extract has medicinal potential against bacterial infection. The human red blood cell

(HRBC) membrane stabilization method was used in the experiment to evaluate in vitro anti-

inflammatory activity, while the disc diffusion method was used to assess in vitro antibacterial

activity using cultures of Escherichia coli and Staphylococcus aureus.

These theories will serve as the researchers' guides as they attempt to verify and learn

more about how to treat Salmonella typhimurium utilizing the disk diffusion method

employing calabash fruit extract and azithromycin.

Statement of the Problem

1. What are the cultural characteristics of Salmonella typhimurium exposed to various

antibiotics of Crescentia cujete extracts based on the following parameters?

1.1 Colony count

1.2 Shape

1.3 Form

1.4 Color

1.5 Margin

2. What is the zone of inhibition of the following Crescentia cujete fruit extracts to the

Salmonella typhimurium using Kirby-Bauer Disk Diffusion Method?

1. 50% Fruit extract

2. 75% Fruit extract

 3. 100% Fruit extract

4. Commercial Antibiotic

3. What is the degree of susceptibility of Salmonella typhimurium to the Crescentia

cujete fruit extract and Azithromycin in Terms of the following parameters:

1. Cultural characteristics

2. Zone of inhibition

Hypothesis

Here are the outcomes the researchers expect from the said experimentation after

undergoing various testing and examination.

1. Calabash as a fruit with high antibacterial substances can stand and eradicate

Salmonella typhimurium.

2. Calabash extract properties are completely treated the Salmonella typhimurium

without observable negative sign.

3. Calabash’s effectiveness can be considered effective based on the Azithromycin

antibiotic.

Scope and Limitations

This study focuses on the effectiveness of Calabash’s Extract specifically its

antibacterial activity in the fight against Salmonella typhimurium which is a pathogen that most

often causes gastroenteritis in humans. The research topic bears significance as the researchers

will try to find out its potentiality and capableness in killing bacteria to broaden its beneficial

gap and expediency in contributing to the medical field.

 For this study, the research will only use the extract that comes from the Calabash Fruit

to test its effectiveness in eradicating Salmonella typhimurium bacteria when in contact with

one another, also together with antibiotics, particularly with Azithromycin that has proven to

kill Salmonella typhimurium and test its similarities on how Calabash’s extract will react the

same to further know its accuracy in terms of its antibacterial effectiveness. Moreover, this

study will not extend its testing material to any kind of living organism specifically to humans

to avoid hazardous consequences and also to ensure no one will be put in harm as the researcher

follows adequate safety guidelines while performing this experimental research.

Furthermore, this experimental study will not be held physically inside Buting Senior

High School for the reason, Salmonella typhimurium is a contagious bacterial illness that can

cause an outbreak or rapid widespread in the community hence, it is better not to perform it

inside the school’s laboratory to avoid and prevent for the worst-case scenario to happen. The

researchers prefer to conduct this experiment with the help of a Biologist or an expert in the

specific branch of science concerning the study of living organisms provided with their testing

facilities for the researcher to have an explicit track to gain comprehensive knowledge and

information to accomplish the said experimental research.

Significance of the Study

In the Philippines, the use of alternative medicine is encouraged by the Philippine

Institute of Traditional and Alternative Health Care (PITAHC), an arm of the Department of

Health whose primary mission is to provide people with healthcare through traditional and

alternative sources of medicine that are proven to be safe, affordable, and effective.
 Thus, this research study was conducted to help the government's objective of

discovering plant sources for the treatment and management of human diseases. Initially, the

study identifies the antibacterial effectiveness of Calabash fruit extract against Salmonella

typhimurium. Moreover, the results of the study will be beneficial to the following:

Medical Field. The result of this study can be a significant endeavor in acquiring additional

knowledge and spreading information on whether the said miracle fruit (C. cujete) has the

antibacterial effect against Salmonella typhimurium.

Government agencies such as the Department of Science and Technology (DOST),

Department of Health (DOH), and Department of Agriculture (DOA). The insights

provided by this study can help such organizations' Research and Development departments.

The development of new or improved initiatives for science and health may increase. Further,

this would enable the production of Calabash in the country to expand given the demands of

scientific and medical research.

Community. As the Calabash (Crescentia cujete) is being grown in a lot of places here in the

Philippines, this study will contribute to developing a safe and effective way that can help rid

of Salmonella typhimurium. Filipinos would be able to utilize a safe and natural alternative in

dealing with such illness.

Future Researchers. This study will aid future researchers in their understanding of the

antibacterial effectiveness of Calabash extract. It will help them obtain more information and
references to assist their Salmonella typhimurium study. They could use this paper as a reliable

source to acquire ideas for their research.

Definition of Terms

For better understanding of the study, the following terms are defined in the context of

this research.

Allopathic. It refers to modern western medicine where healthcare professionals use

scientific research to diagnose and treat symptoms and conditions.

Azithromycin. It is a broad-spectrum macrolide antibiotic that is use to treat and

control the salmonella typhimurium.

Calabash. A tropical fruit presumed to have antibacterial properties that could help

treat salmonella typhimurium

Colony. It is a collection of microorganisms grown on a solid medium that eventually

form a mass for use in laboratories.

Cultural Characteristics. It is the characteristics of a microorganism while being use

in an experiment.

Disc Diffusion. A technique for determining microbial susceptibility to antibiotics in

which filter paper disks containing known antibiotic concentrations are placed on an agar

surface previously inoculated with the bacteria of interest.

Extract. This refers to the preparation of an object containing active ingredients that is

required for experimentation. It is the removal of something from an object by force.

Filter Paper Disk. It is a small circular paper used to collect extract from the sample.

Petri Dish. It is a transparent circular dish with a flat lid used for microorganism

culture.
 Salmonella typhimurium. It is a gram-negative rod Enterobacteriaceae that causes

serious illness in humans. A bacterium being treated with calabash extract.

Zone of Inhibition. It is a circular area around the antibiotic spot where bacteria

colonies do not grow to test the bacteria's susceptibility to the antibiotic.

References:

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2022, from https://www.plantsjournal.com/archives/2019/vol7issue3/PartB/7-3-5-

783.pdf

Institute, N. C. (2020, February 7). Disk diffusion method. Qeios. Retrieved October 17, 2022,

from

https://www.qeios.com/read/YA95FW?fbclid=IwAR10iTe0TWchU4W9CvXEzpxswjl

TIAuVkWOXJM_D9l-NIAcAsnh8IWelq_kKurtz, J. R., Goggins, J. A., &

McLachlan, J. B. (2017, July 15). Salmonella infection: Interplay between the bacteria

and host immune system. Immunology Letters. Retrieved October 16, 2022, from

https://www.sciencedirect.com/science/article/abs/pii/S0165247817302018

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Parvin, M. S., et al., (2015, September 4). Evaluation of in vitro anti-inflammatory and

antibacterial potential of Crescentia cujete leaves and stem bark. BMC research notes.

Retrieved October 16, 2022, from

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Testing.com. (2020, December 18). Antibiotic Susceptibility Testing.

https://www.testing.com/tests/antibiotic-susceptibility-testing/

WebMD. (n.d.). Azithromycin oral: Uses, side effects, interactions, pictures, warnings &

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250-500-mg-oral/details

World Health Organization. (n.d.). Salmonella (non-typhoidal). World Health Organization.

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sheets/detail/salmonella-(non-typhoidal)?fbclid=