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Antibiotic resistance is one among the leading threats to the health

sector worldwide. This phenomenon occurs when bacteria adapt and evolve
in response to the usage of antibiotics. While it is a naturally occurring process
as a result of natural selection, the misuse and extreme overuse of these
medicines, alongside poor prevention and control accelerates antibiotic
resistance and the emergence of many Multi-Drug Resistant Organisms. Cases
of antibiotic resistance has been rising to a threatening level around the globe
where areas with unstable economic conditions and poor healthcare systems
are the most vulnerable.

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Bacteria develop mechanisms to defend against antibiotics through
genetic mutations with selective pressure and horizontal gene transfer.
Moreover, several bacteria have developed enzymes within their cells which
hydrolyze wide selection of antibiotics which then render these drugs
ineffective in combating bacterial infections. Such enzymes include
carbapenemases which is a type of β-lactamase enzyme which hydrolyze
most classes of β-lactam antibiotics including carbapenems.

These antibiotic resistant mechanisms have led to the development of

several drug combinations with novel enzyme inhibitors to extend the
effectivity of present β-lactam antibiotics. β-lactamase inhibitors include
sulbactam, tazobactam, avibactam, vaborbactam, and relebactam are co-
administered in synergy with β-lactams such as carbapenems.
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Meanwhile, there are many plants which possess secondary metabolites
with potential antimicrobial and enzyme inhibition properties.
Scientists have been compelled to study plants in order to identify
potential antimicrobial and enzyme inhibiting properties due to the presence
of various life-sustaining components as well as cheaper options in treating
several ailments including bacterial infections caused by carbapenemase-
forming microorganisms.

Such secondary metabolites include tomatidine which is a steroidal

alkaloid obtained from the stem and leaves of unripe, green tomatoes which
carries many potential therapeutic activities. Thus, this study will be conducted
to determine the potential beta-lactamase inhibition and bactericidal activity
of tomatidine in synergy with carbapenems in carbapenemase-forming gram
negative bacteria

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Generally, this study aims to determine and evaluate the Potential
Beta-Lactamase Inhibition and Bactericidal Activity of Tomatidine in Synergy
with Carbapenems in Carbapenemase-Forming Gram Negative Bacteria
Specifically, this study aims to:

1. Isolate and identify gram-negative bacteria from sampling site

(Iguig Sanitary landfill)
2. Determine the presence of carbapenemases in isolated gram-
negative bacteria.
3. Evaluate the carbapenemase inhibiting activity of tomatidine in
gram negative bacteria
4. To evaluate the bactericidal activity of tomatidine in synergy with
carbapenems against Carbapenemase-forming Gram Negative
Bacteria isolates at different % concentrations.
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This study will focus on the potential potential beta-lactamase inhibition
and bactericidal activity of tomatidine in synergy with carbapenems in
carbapenemase-forming gram negative bacteria. Tomatidine compound will
be purchased from a specific supplier for minimal error on isolation procedures.

Isolates will only be isolated from the garbage, soil, and water samples
form Iguig Sanitary Landfill alone.
The study will focus only on enzyme inhibition against carbapenemases.
Purification and genotypic identification of the specific carbapenemase will
not be conducted. Antibiotic studies will only focus on carbapenems including
Imipenem, Doripenem, Ertapenem, and Meropenem. Formulation of potential
capsule product and other drugs will not be conducted in the study.

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This study will be of huge significant to the following group of people:
1. Public
2. Community

3. Department of Health
4. Department of Science and Technology
5. Future Researchers
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The study will be conducted on the month of November 2023 at the
Department of Agriculture (DA), Department of Science and Technology
(DOST), Carig Regional Center, Carig Sur, and CSU Central Laboratory
Tuguegarao City.

8. Experimental Design
This study will be using an experimental research design that will involve
a control the purpose of studying the Beta-lactamase inhibition and
bactericidal activity of tomatidine in synergy with carbapenems in
carbapenemase-forming. This research study will lay out Completely
Randomized Design (CRD).
9. Collection, Isolation, and Identification of Bacterial Samples

Bacterial samples will be obtained from the wastes and soil samples in
Iguig Sanitary Landfill. Bacterial isolates will be grown in MacConkey Agar
(MAC) which is a selective media for Gram-Negative Bacteria. Isolates will be
incubated for 24 hours before subjecting to purification by inoculating different
colonies and growing them in separate MAC plate. Bacteria grown in the
medium will be subjected for biochemical testing using VITEK for species
identification. Target species to be identified are Escherichia coli, Klebsiella
pneumoniae, and Pseudomonas aeruginosa.

10. Antimicrobial Susceptibility Testing adopted from CLSI (2020)

Pure culture isolates will be subjected to antimicrobial susceptibility

testing by determining their Minimum Inhibitory Concentration (MIC) by broth
microdilution technique in Cation-adjusted Mueller Hinton Broth (CAHMB).
11. Test for Carbapenemases
Isolates positive for antimicrobial resistance or are not susceptible to one
or more carbapenems will be subjected to Test for Carbapenemases using
CarbaNP Test following CLSI recommendation. Researchers will be utilizing
RAPIDEC Test Kit to test each isolate for rapid detection of Carbapenemase.
Interpretative categories will be based on product guidelines.

Acquisition of Tomatidine Compound

Tomatidine compound will be acquired from Enzo Life Sciences Inc.

Tomatidine will be solubilized in DMSO at 2 mg/ ml.

12. Carbapenemase Isolation and Purification adopted from Neu et. al (1978)
The carbapenemases will consist of bacterial cells extracted via sonic
treatment. Each isolate will be grown from a single colony inoculum into
nutrient agar at neutral pH containing 20 µg/ml imipenem as inducer for
production of the enzyme and will be incubated for three hours. The bacterial
cells will be collected by centrifugation, resuspended, and will be washed
using phosphate buffer. The cells will be disrupted through sonic disintegration
for 5 minutes in an ice bath. Cells will be subjected to centrifugation for two
hours at 40,000 x g at 4⁰C to remove cellular debris. Supernatant material will
undergo dialysis against phosphate buffer for 24 hours at 4⁰C.
13. Carbapenemase Assay and Inhibition adopted from Neu et. al (1978)
Carbapenemase activity will be identified through UV-visible
spectrophotometric method by change in optical density at 300 nm with
imipenem as substrate. The same method will be followed for the observation
of the inhibition of imipenem hydrolysis by incubating carbapenemase with
tomatidine before the addition of substrate.

14. Bactericidal and Synergy Test adopted from Mitchell et. al (2011)
Synergistic activity of tomatidine will be identified following
checkerboard protocol by a broth microdilution method in order to evaluate
the effect of tomatidine at various concentrations on the activity of
carbapenems against carbapenemase-producing gram negative bacteria