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Pontifical and Royal

UNIVERSITY OF SANTO TOMAS


THE CATHOLIC UNIVERSITY OF THE PHILIPPINES

Senior High School


Capsule Research Proposal Form – HA Strand
Proponents: Agustin, Ellana Mickaela Jao
Chua, Sophia Angelique Bobis
Eduarte, Mikhaela Genevieve Gonzales
Macabangon, Eunice Contreras
Soriano, Janelle Rosario
Villafuerte, Miko Bosito
Potential of Pharaoh Cuttlefish (Sepia pharaonis) Ink as β-lactamase Inhibitor
Project Title:
Against Multidrug-Resistant Mycobacterium tuberculosis

Project Discipline The University of Santo Tomas have five research flagships namely:
Cluster (Shade all that apply): □ Science and Technology
□ Health & Allied Sciences
□ Social Sciences and Education
□ Arts and Humanities
□ Sacred Sciences

Among the areas of research in the university are:


□ Natural products □ Climate Change Preparedness
□ Chemical Sensors □ Education
□ Biosensors □ Arts
□ Materials Science □ Culture
□ Energy research □ Humanities
□ Pure and Applied Microbiology □ Gerontology
□ Biodiversity □ Migration Studies
□ Environmental Science □ Youth Studies
□ Food Science and Engineering □ Corporate Social Responsibility
□ Urban Environment □ Other, specify: _________________________

Proposal Abstract: (Briefly summarize objectives, issues to be addressed, methods, and expected output. Min-Max no. of words = 150-200)
Tuberculosis has been infecting humans for many decades and is still considered as a
modern day problem. The increase of multidrug resistance of bacteria at a terrifying rate has
been the major cause of the intensifying rate of TB morbidity.

AAYUSIN KO PA TO TEKA LANG

Objectives: (Indicate general and specific objectives that needs to be addressed by the project. Min-Max no. of words = 30-100)
Declared as one of the deadliest infectious disease by the World Health Organization, the
emergence of the drug resistance of tuberculosis made it more difficult to find a way to
eradicate it.
This study will pave way to a solution regarding today’s major concern by finding an
alternative way to prevent the bacteria Mycobacterium tuberculosis from desisting antibiotics.
This research doesn’t just aim to determine the properties of sepia ink but to specifically
prove the potential of Pharaoh cuttlefish (Sepia pharaonis) as β-lactamase inhibitor to prevent
the resistance of the M. tuberculosis.

Significance: (State the issues the research aims to address, and the significance of this research project in relation to other researches
on the same topics/ consider social and technological impact of the research project. Min-Max no. of words = 50-100)
Mycobacterium tuberculosis is the causative agent that resists several drugs and yields the
disease called tuberculosis. The researchers believe that this study would have a major
contribution to the field of microbiology through the use of the results gained against the
multidrug resistance of the bacteria. It will also help the researchers find solutions and discover
drugs most especially for those who are affected by this disease. This research would
strengthen other studies leading to the advancement of drug discoveries and would find other
unknown properties of the variable to be used in this research that are naturally occurring.
Literature (Related works or studies in the research area. Min-Max no. of words = 800-1000)
Review: Declared as a global emergency by the World Health Organization (WHO) in 1993,
Tuberculosis remains as one of the leading health problems worldwide (Da Silva, P. E. A. et al.,
2011). It is considered as a significant infectious disease because even in first world countries
with extensive control programs, it spreads like wildfire (Gardy, J. L. et al., 2011). According to
WHO in 2015, 10.4 million people were diagnosed with the disease, among which 1.8 million
passed away (Nguyen, H.Q. et al., 2017). This sickness is presently treated with a particular
recommended program of medication by combining isoniazid, pyrazinamide, rifampicin and
ethambutol. Research seems to agree that the use of prominent drugs integrated with the
classic drugs could be a good alternative treatment (Pagliotto, A.D.F et al., 2015). It has been
found that failure to supply sufficient medication and patient’s refusal to comply with the
treatment can lead to the resistance of its bacterial pathogen, Mycobacterium tuberculosis.

Drug resistance serves as a serious threat to the overall global health conditions. For the
past decade, reported cases of drug-sensitive tuberculosis have greatly decreased. However,
this improvement is crucially weakened by the rise of multidrug-resistant (MDR) and extensively
drug-resistant (XDR) tuberculosis. Multidrug-resistant tuberculosis is brought by
Mycobacterium tuberculosis and is resistant to first-line drugs for combating tuberculosis,
specifically isoniazid and rifampicin; while extensively drug-resistant tuberculosis is defined as
multidrug-resistant tuberculosis with additional resistance to fluroquinolone and at least one of
the three inoculated medications: kanamycin, amikacin, and capreomycin (Gandhi, N. R. et al.,
2010).

Early diagnosis, proper adherence to therapy, and drug development are important
factors that need to be considered to satisfy the challenges brought by MDR and XDR (Zhang, Y.
et al., 2004). WHO acknowledged that the pursuit for finding a cure for drug-resistant
tuberculosis is one of the most significant challenges concerning global health (Nguyen, H. Q. et
al., 2017). In 2008, based on a study done by the same organization, the number of cases of
drug-resistant tuberculosis worldwide has reached an ultimate high, making all attempts to
solve the problem almost useless (Jassal, M. et al., 2009). Treating MDR and XDR is much more
challenging than treating drug-susceptible tuberculosis because it costs more than usual
treatment prices, and also requires more time and effort, making it more prone to failure and
even death (Gandhi, N. R. et al., 2010).

β-lactam antibiotics are the most commonly used antibiotics worldwide due to their
notable antimicrobial properties designed to destroy microorganisms. It prevents the
transpeptidase and peptidoglycan strands from conjoining together resulting in the inhibition of
the cell wall biosynthesis (Liu, X-L. et. al., 2016). Bacteria resist these drugs due to the irregular
and excessive use of antimicrobial products to ensure their survival by producing β -lactamase
enzymes. This is the most basic and effectual way to destroy B-lactam antibiotics (Haq, F. U. et.
al, 2017).

β -lactamases are enzymes that are manufactured and released by certain bacteria,
primarily causing its resistance to the corresponding β -lactam antibiotics targeting it. This
protective mechanism is a result of the self-defense of bacteria against the environment or a
contribution to the physiological role of the assembly of peptidoglycan. The breaking of the
amide bond of the lactam ring, which is the core structure of the antibiotic, and the C-N bond,
which is in the four-membered ring that would undergo hydrolysis, conducted by these
enzymes will result in the ineffectiveness of the antibiotic (Haq, F. U. et. al, 2017; Liu X-L. et. al.,
2016). Hence, more studies were conducted prior to the conclusion that bacterial resistance
was in need of increased attention as it is an arising global threat to the health of people.

One bacterium found to possess resistance to β -lactam antibiotics was M. tuberculosis. Its
production of enzyme β -lactamase served as the primary cause of its ability to resist the
execution process of the drug (Dincer, I. et al., 2004). The amount of enzyme β -lactamase to
be liberated in a culture is dependent on the growth condition and strain of the bacteria; and
that such factors are likely to have increased their effectivity when in a salty environment
(McDougal, L. K. et al., 1986).

Being among the dominant predators in the ocean that are also solely marine,
cephalopods are intelligent species and possess a lot of medicinal properties (Shiva Shakthi, S.
et al., 2016). In 2001, the production of cephalopods in significant fishing areas around the
world projected 3346.8 metric tonnes; wherein 16% of these were cuttlefish (Vaz-Pires, P. et al.,
2008). The most abundant in the region of Indo-West Pacific is the Pharoah Cuttlefish (Sepia
pharaonis), which makes them superior among other species in the marketable fish production
(Al Marzouq, A. et al., 2009). Pharoah cuttlefish is known for its high density cultivation due to
some of its characteristics like high feed conversion rate, high protein content, and rapid growth
rate (Wen, J. et al., 2016). Various studies have found that cephalopods’ most notable feature is
their capability to eject ink caused by external stimuli to respond against predators and other
threats by emerging mechanisms (Wen, J. et al., 2016; Shiva, Shakthi S. et al., 2016).

Long known for its efficacy in promoting human health, cephalopod inks are currently
being used as assays to create novel drugs by looking for natural products containing medicinal
properties (Derby, C., 2014). Cephalopods’ inks could increase nonspecific immune response
and leukocytes, possess capability of blood congealment, and contain anti-cancer properties. It
also contains a lot of therapeutic properties such as antimicrobial, anticancer, anti-
inflammatory; enormous prospects in various diseases like uterine dysfunction; and even
hepatoprotective and haemopoetic effects (Chisti, M. R., et al.) Since the ink has been a waste
material of processing industry, its cost and availability is of no problem. Its convenient
accessibility can possibly pave way to a lot of studies like the potential of this raw material for
drug production (Shiva Shakthi, S. et al., 2016).

Methodology: (Provide a schematic diagram of the data collection procedures/experiment, including materials for each process.)
I. Collection and Preparation of Ink Samples
II. Acquisition and Preparation of the bacteria Mycobacterium smegmatis

III. Preparation and Activation of β-lactamase


IV. Screening of the ink extract for β-lactamase inhibition

V. Analysis of Data

Target Beneficiaries: (Identify the clienteles or recipients who will benefit from the research project. Min-Max no of words: 20-100)
This study will benefit researchers that are of the same field as the study. Findings yielded
will add to the existing body of knowledge in microbiology, which could also be a source of new
ways in order to form methods and techniques in addressing drug resistance and even
tuberculosis.

The study will also be able to benefit patients affected by the disease. This may bring forth
to the advancement in the effectivity of medicine they use in response to Tuberculosis, leading
to a shorter amount of time for recovery.

Expected Outputs: (Indicate the specific products, processes, and results which the project is expected to produce and how these outputs
may be used. Min-Max no. of words: 30-100)
After the experiment, the researchers would find inhibitory properties from the Sepia ink
against β-lactamase produced by Mycobacterium Tuberculosis. It can be used as a β-lactamase
inhibitor in order to increase the effectivity of β-lactam antibiotics.

The Pharaoh cuttlefish ink will be discovered to possess a lot of components that are
useful in the field of science. The inhibitory property of its ink will be used by researches within
the similar field of study in the future. It will contribute to drug development and formulation
for multi-resistant bacteria such as Mycobacterium Tuberculosis.
References: (Make a reference list using APA format 6th edition.)
Akkiraju, L. J., Sowmya, M., Deepthi, Ch., & Lakshmi, V. V. (2015). Herbal

extracts as beta lactamase inhibitors. Internation Journal of Scientific &

Engineering Research, 6, 68-70.

Al Marzouqi, A., Jayabalan, N., & Al-Nahdi, A. (2009). Biology and stock

assessment of the pharaoh cuttlefish, Sepia pharaonis ehrenberg, 1831

from the arabian sea off oman. Indian Journal of Fisheries, 56, 231-239.

Chisti, M. R., Raza, A., Singh, P. A., Niazi, J. (2016). Sepia Ink: An untouched

molecule from deep oceans. International Journal of Research and

Reviews in Pharmacy and Applied Sciences, 6, 1303-1307.

Da Silva, P. E. A., & Palomino, J. C. (2011). Molecular basis and mechanisms

of drug resistance in Mycobacterium tuberculosis: classical and new

drugs. Journal of Antimicrobial and Chemotherapy, 66, 1417-1430.

Derby, C. (2014). Cephalopod ink: production, chemistry, functions and

applications. Marine Drugs, 12, 2700-2730.

Dincer, I., Ergin, A., & Kocagöz, T. (2003). The vitro efficacy of β- lactam and

β-lactamase inhibitors against multidrug resistant clinical strains of

Mycobacterium tuberculosis. International Journal of Antimicrobial

Agents, 23, 408-411.

Gandhi, N. R., Nunn, P., Dheda, K., Schaaf, H. S., Zignol, M., Sooligen, D. V. .

. . Bayona, J. (2010). Multidrug- resistant and extensively drug-resistant

tuberculosis; a threat to global control of tuberculosis. The Lancet, 375,

1830-1843.

Gardy, J. L., Johnston, J. C., Ho Sui, S. J., Cook, V. J., Shah, L., Brodkin, E., ...

Tang, P. (2011). Whole-genome sequencing and social network analysis

of a tuberculosis outbreak. The New England Journal of Medicine, 364,

730-739.

Haq, F. U., Abro, A., Raza, S., Liedl, K. R., & Azam, S. S. (2017). Molecular
dynamics simulation studies of novel β-lactamase inhibitor. Journal of

Molecular Graphics and Modelling, 74, 143- 152.

Jassal, M., & Bishai W. R. (2009). Extensively drug-resistant tuberculosis. The

Lancet infectious diseases 2009, 9, 19-30.

Liu, X-L., Yang, K-W., Zhang, Y-J., Ge, Y., Xiang, Y., Chang, Y-N., &

Oelschlaeger, P. (2016). Optimization of amino acids thioesters as

inhibitors of metallo-β-lactamase L1. Bioorganic & Medicinal

Chemistry Letters, 26, 4698-4701.

McDougal, L.K., & Thornsberry, C. (1986). The role of β-lactamase in

staphylococcal resistance to penicillinase-resistant penicillins and

cephalosporins. Journal of Clinical Microbiology, 23, 832- 839.

Nguyen, H. Q., Nguyen, N. V., Contamin, L., Tran, T. T., H., Vu, T. T.,

Nguyen, H. V., ... Nguyen, V. A. T. (2017). Quadruple-first line drug

resistance in Mycobacterium tuberculosis in Vietnam: What can we

learn from genes?. Infection, Genetics, and Evolution, 50, 55-61.

Pagliotto, A. D. F., Caleffi-Ferracioli, K. R., Lopes, M. A., Baldin, V. P., Leiti,

C. Q. F., Pavan, F. R., ... Cardoso, R. F. (2016). AntiMycobacterium

tuberculosis activity of antituberculosis drugs and

amoxicillin/clavulanate combination. Journal of Microbiology,

Immunology, and Infection, 49, 980-983.

Shiva Shakthi, S., Archana, S. P., Arulvasu, C., & Neethiselvan, N. (2016).

Biochemical composition of cuttle fish Sepia prabahari ink and its

bioactive properties in-vitro. International Journal of Pharmaceutical

Sciences and Research, 7, 2966-2975.

Smiline, G. A. S., Vijayshree, P. J., Pandi, S. K., & Raguraman, R. (2011).

Antibacterial effect of squid ink esbl producing strains of Escherichia

coli and Klebsiella pneumoniae. Indian Journal of Geo-Marine


Sciences, 41, 338-343.

Vaz-Pires, P., Seixas, P., Mota, M., Lapa-Guimarāes, J., Pickova, J., Lindo, &

A., Silva, T. (2008). Sensory, microbiological, physical and chemical

properties of cuttlefish (Sepia officinalis) and broadtail shortfin squid

(Illex coindetii) stored in ice. LWT – Food Science and Technology, 41,

1655-1664.

Wen, J., Zhong, H., Xiao, J., Zhou, Y., Chen, Z., Zeng, L., ... Wang, F. (2016).

A transcriptome resource for pharaoh cuttlefish (Sepia pharaonis) after

ink ejection by brief pressing. Marine Genomics, 28, 53-56.

Zhang, Y., & Yew. W. W. (2004). Mechanisms of drug resistance in

Mycobacterium tuberculosis. Frontiers in Bioscience, 9, 975- 994.

Note: Table may be adjusted to accommodate entries.

BUDGETARY REQUIREMENTS
ITEMS AMOUNT (in PHP) REMARKS
A. Materials
- Sepia pharaonis Php 1,250 5 kg
- Mycobacterium smegmatis Php 13, 200 3 ampoules - freeze dried
- Surgical gloves Php 150 1 box
- Surgical Mask Php 75 1 box
- Laboratory Gown Php 900 30 disposable lab gowns
- Beaker Php 1,200 2 beakers
- Syringe (5) Php 2,000 2 stainless syringe 50 cc
- Glass Stirring Rods Php 360 3 stirring rods
- Tweezers Php 446 6 in 1 set
- Wooden Rack Php 120 1 rack
- Nutrient Agar Php 2,405 100 g
- Nutrient Broth Php 2,014 100 g
- Amber bottle Php 100 4 bottles
- Pipette Php 280 1 pc.
- Petri dish Php 300 3 pcs.
- Whatman Filter Paper Php 1,500 1 box
- Hexane Php 600 1 bottle
- Penicillin (Liquid form) Php 168 1 bottle
- Starch Php 70 1 bottle
- Iodine Solution Php 280 1 bottle
B. Services (Lab testing fees, etc.)
- Heidolph, VE-11 Rota Evaporator Php 2,699
- Marine Microbiologist expert Php 5,000
- Laboratory use Php 15,000
C. Others (Please specify)
- Transporation Php 480 UST – Pasay City and vice versa
-
TOTAL Php 50,597.
Note: Table may be adjusted to accommodate entries.
WORK PLAN
ACTIVITIES IMPLEMENTATION DATE OUTPUT
Purchasing of Bacteria Ampoules of Obtaining the bacteria,
August 12-September 2
M. smegmatis M. smegmatis
Purchasing of Laboratory Equipment August 12-19 Obtaining Laboratory Equipment
Purchasing of Pharaoh Cuttlefish and Extraction of the Ink from the
August 20-27
Preparation of the crude ink extracts obtained pharaoh cuttlefish
Culturing of the bacteria, M. smegmatis September 2-3 Growth of bacteria
Presence of the activated β-
Activation of β-lactamase September 3
lactamase
Identification of sepia ink’s inhibitory Sepia ink shows inhibitory
September 3-4
properties through MIC test properties against β-lactamase
Analysis of data and results September 4-10 Gaining the results
Finalization of the research paper September 10-October 10 Complete paper
Note: Table may be adjusted to accommodate entries.

PROFILE OF THE RESEARCHERS


Researcher 1
A. Personal information
Name Agustin, Ellana Mickaela Jao
Age 16
Sex Female
Birthdate December 18, 2000
Mailing Address #1237 Antonio St., Moradiane Park, Sampaloc, Manila
E-mail Address mickaagustin910@gmail.com
Mobile No. 09164624918
Telephone No. None
B. Educational Background
Elementary Cauayan South Central School
Secondary (JHS) Our Lady of the Pillar College-Cauayan City, Isabela
Secondary (SHS) University of Santo Tomas
C. Research-related 2015 Philippine Association of Chemistry Teachers, Inc.- Region III Pact III
engagements (past research Annual Seminar Workshop
works, awards, trainings, etc; Junior Investigatory Project
enumerate all.) Our Lady of the Pillar College-Cauayan Research Seminar 2015
Researcher 2
A. Personal information
Name Chua, Sophia Angelique Bobis
Age 17
Sex Female
Birthdate August 24, 1999
Mailing Address Lot 18c, Blk. 2, L. Santos St., Brgy. San Juan, Cainta, Rizal
E-mail Address imsophiachua@gmail.com
Mobile No. 09178034577
Telephone No. 656-8107
B. Educational Background
Elementary St. Francis of Assisi Montessori School of Cainta
Secondary (JHS) St. Francis of Assisi Montessori School of Cainta
Secondary (SHS) University of Santo Tomas
C. Research-related
engagements (past research None
works, awards, trainings, etc;
enumerate all.)

Researcher 3
D. Personal information
Name Eduarte, Mikhaela Genevieve Gonzales
Age 17
Sex Female
Birthdate October 2, 1999
Mailing Address Blk 14 Lot 31 Lagro Subd., Fairview, Q.C.
E-mail Address mge100299@yahoo.com
Mobile No. 09166475594
Telephone No. 7011601
E. Educational Background
Elementary Little Merry Hearts Montessori Center Q.C.
Secondary (JHS) Little Merry Hearts Montessori Center Q.C.
Secondary (SHS) University of Santo Tomas
F. Research-related
engagements (past research None
works, awards, trainings, etc;
enumerate all.)
Researcher 4
G. Personal information
Name Macabangon, Eunice Contreras
Age 17
Sex Female
Birthdate June 13, 1999
Mailing Address Blk. 5 Lt. 3, Grand Royale Subdv., Bulihan, Malolos, Bulacan
E-mail Address eunicemacabangon@gmail.com
Mobile No. 09266778187
Telephone No. None
H. Educational Background
Elementary Holy Spirit Academy of Malolos
Secondary (JHS) Holy Spirit Academy of Malolos
Secondary (SHS) University of Santo Tomas
I. Research-related
engagements (past research None
works, awards, trainings, etc;
enumerate all.)
Researcher 5
J. Personal information
Name Soriano, Janelle Rosario
Age 18
Sex Female
Birthdate April 18, 1999
Mailing Address Sun Residences, Welcome Rotonda, Quezon City
E-mail Address soriano_janelle18@yahoo.com
Mobile No. 09153803136
Telephone No. None
K. Educational Background
Elementary Mother Goose Special School System, Inc.
Secondary (JHS) Mother Goose Special Science High School
Secondary (SHS) University of Santo Tomas
L. Research-related
engagements (past research Grade 10 Investigatory project entitled: “Eggshells and latex of jackfruit
works, awards, trainings, etc; (Artocarpus heterophyllus) as an alternative roof sealant”
enumerate all.)
Researcher 6
M. Personal information
Name Villafuerte, Miko Bosito
Age 16
Sex Male
Birthdate August 18, 2000
Mailing Address G. Tolentino St., Sampaloc Manila
E-mail Address mikovillafuerte88@gmail.com
Mobile No. 09083832277/09290870770
Telephone No. None
N. Educational Background
Elementary Siena College Tigaon
Secondary (JHS) Siena College Tigaon
Secondary (SHS) University of Santo Tomas
Grade 10 Research paper entitled: “Factors Affecting Students’
O. Research-related Concentration
engagements (past research Students of Siena College Tigaon
works, awards, trainings, etc;
enumerate all.) S/Y 2015-2016”

Note: Table may be adjusted to accommodate entries.


MR. KIN ISRAEL NOTARTE
Research Teacher
Signature over Printed Name