A Comparison of the In-Vitro Bacterial Growth Inhibition Properties of Grape Seed

Extract and Antibiotics When Tested Against Staphylococcus aureus, Escherichia

coli, Pseudomonas aeruginosa, and Bacillus subtilis Bacteria

Allison Burritt

A Thesis Presented to the Central Magnet Faculty in Partial Fulfillment of the

Graduation Requirements

May 2023
 Abstract

The overuse and misuse of antibiotics since their origin in 1928 has led to the
development of antibiotic-resistant bacteria. The creation of new drugs is costly and
limited, so the rapid emergence and spread of antibiotic-resistant bacterial strains has
emphasized the need for alternative antibacterial agents that act through new mechanisms
of bacterial inhibition. The purpose of this study was to compare the bacterial inhibition
properties of grape seed extract, penicillin, tetracycline, ciprofloxacin, tobramycin,
sulfamethoxazole/trimethoprim, and polymyxin B when tested against Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis to determine the
viability of grape seed extract as an alternative antibiotic treatment. The Kirby Bauer disc
diffusion method was used to determine the susceptibility of the bacteria to the chosen
antibacterial agents. Ultimately, grape seed extract only inhibited the growth of S. aureus
and had zero inhibitory effect on E. coli, P. aeruginosa, and B. subtilis. However, grape
seed extract proved to be more effective in inhibiting the growth of S. aureus than
penicillin and polymyxin B as the bacterium was resistant to both of these antibiotics.
Grape seed extract’s results were promising, and further research into its abilities could
make it an effective alternative antibacterial treatment in the future.
 Acknowledgements

I would like to express my sincere gratitude to my mentor, Dr. John Zamora. He

generously provided me with my needed materials, taught me the methodology to carry

out my research, and allowed me to use his workspace to perform my experiment. I

would also like to thank Mrs. Eve Harrison, my field of study advisor. Her guidance and

advice throughout my research were invaluable. Finally, I would like to thank Dr. Laura

Lynn Roland for her tremendous support over the duration of this research project. I

couldn’t have done it without them.

 Table of Contents

List of Tables.....................................................................................................................vi

List of Figures...................................................................................................................vi

Chapter 1: INTRODUCTION..........................................................................................1

Research Question..........................................................................................................1

Background Information...............................................................................................1

Definition of Terms........................................................................................................2

Purpose of Study............................................................................................................3

Hypothesis.......................................................................................................................3

Chapter 2: REVIEW OF LITERATURE.......................................................................4

Effectiveness of Grape Seed Extract as an Antibacterial Agent................................4

Methicillin-Resistant Staphylococcus aureus..............................................................5

Phenolic Content of Grape Seed Extract.....................................................................5

Gallic Acid...................................................................................................................6

Catechins and Epicatechin...........................................................................................6

Action Mechanisms of Antibiotics................................................................................7

Beta-lactams.................................................................................................................7

Tetracyclines................................................................................................................7

Fluoroquinolones.........................................................................................................7

Aminoglycosides...........................................................................................................7

Macrolides...................................................................................................................8

Sulfonamides................................................................................................................8
 Conclusion.......................................................................................................................8

Chapter 3: METHODOLOGY.........................................................................................9

Overview.........................................................................................................................9

Apparatus and Materials...............................................................................................9

Procedures....................................................................................................................10

Disc Preparation........................................................................................................10

Experimental Procedure............................................................................................11

Data Analysis................................................................................................................11

Chapter 4: FINDINGS AND DISCUSSION.................................................................12

Findings.........................................................................................................................12

Data Collected...........................................................................................................12

Analysis of Data.........................................................................................................13

Discussion......................................................................................................................19

Chapter 5: CONCLUSION.............................................................................................22

Study Summary............................................................................................................22

Limitations....................................................................................................................22

Implications..................................................................................................................23

Suggestions for Future Study......................................................................................24

References.........................................................................................................................25
 List of Tables

Table 1, Average Zone of Inhibition Measurements for Antibacterial Agents..................12

Table 2, Reference Chart to Determine Susceptibility to Select Antibiotics (Adapted from

BBL™ Sensi Disc ™ Antimicrobial Test Discs, July 2011)..............................................13

Table 3, Average Zone of Inhibition Measurements for Antibacterial Agents..................18

List of Figures

Figure 1, Staphylococcus aureus Plates............................................................................14

Figure 2, Bacillus subtilis Plates.......................................................................................15

Figure 3, Escherichia coli Plates.......................................................................................16

Figure 4, Pseudomonas aeruginosa Plates........................................................................17

 Chapter 1: INTRODUCTION

Research Question

How does the in-vitro bacterial growth inhibition of grape seed extract compare to

those of penicillin, tetracycline, ciprofloxacin, tobramycin, sulfamethoxazole/

trimethoprim, and polymyxin B when tested against Staphylococcus aureus, Escherichia

coli, Pseudomonas aeruginosa, and Bacillus subtilis?

Background Information

Grape seed extract is derived from the seeds of Vitis vinifera, a species of grape

commonly used to make wine. Grapes and their seeds have been used as medicinal agents

for thousands of years, dating back to the ancient Egyptians. More recently, grape seed

extract has been praised for its anti-inflammatory and antioxidant properties, so it is often

ingested as an herbal supplement in capsule form (Sego, 2010). In addition, prior studies

have been conducted to determine the effectiveness of grape seed extract as an

antibacterial agent. Though these studies have determined that grape seed extract exhibits

significant bacterial growth inhibition properties, none have been conducted to compare

the effectiveness of these properties to that of antibiotics in order to determine the

viability of grape seed extract as an antibiotic treatment.

The overuse and misuse of antibiotics since their origin in 1928 has led to the

development of antibiotic-resistant bacteria. Bacteria are classified into two categories:

gram-positive and gram-negative. Gram-positive bacteria are distinguished by a cell wall

consisting of a thick layer of peptidoglycan and no outer membrane. Gram-negative

bacteria are distinguished by a cell wall consisting of a thin layer of peptidoglycan and an

outer lipid membrane. The main component of the outer membrane of gram-negative

1
bacteria—lipopolysaccharide—prevents the diffusion of hydrophobic antibiotics across

the membrane, thus preventing antibiotics from targeting the peptidoglycan layer and

working effectively (Breijyeh et al., 2020).

Grape seed extract inhibits the bacterial growth of a variety of species of gram-

positive bacteria, including Staphylococcus aureus, Methicillin-resistant Staphylococcus

aureus, and Bacillus subtilis, as well as a variety of species of gram-negative bacteria,

including Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli

(Alkhulaif et al., 2017); thus, it is well established that grape seed extract inhibits the

growth of gram-negative bacteria, a task that proves to be difficult for traditional

antibiotics. The components of grape seed extract that produce bacteriostatic and

bactericidal effects are phenolic compounds. Polyphenols, which are partially

hydrophobic, decrease the stability of bacterial cell membranes by interacting with the

lipopolysaccharides within the cell wall (Shrestha et al., 2012). Prior research has also

shown that grape seed extract works synergistically with antibiotics to inhibit bacterial

growth (Miklasińska-Majdanik et al., 2018). Not only can grape seed extract effectively

inhibit the growth of several species of gram-positive and gram-negative bacteria, but it

can also work in conjunction with antibiotics to produce an inhibitory response.

Definition of Terms

The following terms will be used throughout various chapters in this research:

 Lipopolysaccharide- a component of the outer membrane of gram-negative

bacteria that serves as a barrier to small, hydrophobic molecules such as

antibiotics and provides structural support for the cell (Bertani et al. 2018)
  Peptidoglycan- a component of most bacterial cell walls that maintains the

structure and shape of the cell and aids in cell growth and division (Vollmer et al.,

2008)

 Polyphenols- a class of compounds derived from plants that include phenolic

acids, flavonoids, lignans, and stilbenes (Pandey et al., 2009)

Purpose of Study

The purpose of this study was to determine the viability of grape seed extract as

an alternative treatment for bacterial infections. The rapid emergence and spread of

antibiotic-resistant bacterial strains has emphasized the need for alternative antibacterial

agents that act through new mechanisms of bacterial inhibition. Through previous

research, it has been established that grape seed extract effectively inhibits the growth of

many common bacteria, so the goal of this study was to compare the inhibitory properties

of grape seed extract to that of several traditional antibiotics so as to establish grape seed

extract as a viable alternative treatment for antibiotic-resistant bacterial infections.

Hypothesis

The bacterial inhibition properties of grape seed extract will be stronger than that

of antibiotics when tested against Escherichia coli and Pseudomonas aeruginosa.

However, the bacterial inhibition properties of grape seed extract will be weaker than that

of antibiotics when tested against Bacillus subtilis and Staphylococcus aureus.

 Chapter 2: REVIEW OF LITERATURE

Effectiveness of Grape Seed Extract as an Antibacterial Agent

Bacteria naturally develop resistance to antibiotics, but humanity’s misuse and

overuse of antibiotics since their inception in 1928 has accelerated this process. Many

strains of bacteria have developed the mechanisms necessary to resist the inhibitory

effects of antibiotics. Consequently, the excessive use of antibiotics has led to the

formation of superbugs. The logical solution to this problem is the development of an

alternative treatment, and several studies introduce grape seed extract as a promising

answer.

Grape seed extract has, in previous studies, exhibited strong antibacterial

properties against both gram-positive and gram-negative samples. In a study on the

antibacterial effect of grape seed extract on susceptible and antibiotic-resistant bacteria, a

clear zone of growth inhibition was observed for all thirty-five gram-positive and gram-

negative bacteria samples, with the clinical isolates showing greater susceptibility to the

extract and Escherichia coli showing the highest susceptibility (Kandasamy et al., 2016).

However, a study on the antibacterial activity of grape seed extract found that of the

tested bacterial isolates, E. coli was least susceptible to grape seed extract (Alkhulaifi et

al., 2017). This discrepancy may come as a result of the variation in solvents used by the

researchers as Kandasamy used dimethyl sulfoxide, and Alkhulaifi used water, ethanol,

and methanol. Nevertheless, grape seed extract has repeatedly been proven to be effective

in the growth inhibition of antibiotic-resistant and susceptible strains of gram-positive

and gram-negative bacteria.

Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a highly transmissible

gram-positive bacterium that is more difficult to treat than other common bacteria due to

its resistance to the beta-lactam class of antibiotics. Extremely prevalent in hospitals,

MRSA often infects people with already weakened immune systems, oftentimes leading

to serious infections and death (Gould, 2005). Although the increasing prevalence of

antibiotic-resistant bacteria is concerning, research on grape seed extract’s ability to

inhibit the growth of MRSA has been promising. In fact, in a study on the bactericidal

effect of grape seed extract on MRSA, forty-three MRSA strains showed sensitivity to

grape seed extract, and 11 of those strains exhibited high sensitivity (Al-Habib et al.,

2010).

Grape seed extract can also be used synergistically with synthetic antibiotics to

produce a stronger inhibitory effect. When used in conjunction with penicillin,

epigallocatechin gallate—a polyphenolic compound found in the seeds of Vitis vinifera—

exhibits a synergistic effect, enhancing the effectiveness of beta-lactam antibiotics

against MRSA. By directly binding to peptidoglycan, epigallocatechin gallate interferes

with the structure and biosynthesis of the bacterial cell wall (Zhao et al., 2002). When

used alone, beta-lactams are ineffective in inhibiting the growth of MRSA, but when used

in conjunction with grape seed extract, the extract enhances the antibacterial activity of

beta-lactams, thus increasing their effectiveness against MRSA.

Phenolic Content of Grape Seed Extract

Several previous studies have established that grapes contain large amounts of

phenolic compounds. More specifically, 60-70% of the total phenolic compounds found
in grapes are located in their seeds (Badet, 2011). Of these phenolic compounds, the most

abundant are gallic acid, catechin, and epicatechin (Sochorova et al., 2020).

Gallic Acid

Gallic acid is exceedingly abundant in the plant kingdom, and it is the most

abundant polyphenol in grape seed extract. This phenolic compound inhibits the growth

of bacteria by disrupting the integrity of the cell membrane. In particular, gallic acid can

change the hydrophobicity, permeability, and charge of the cell membrane (Kahkeshani

et al., 2019). Gallic acid promotes the damage of bacterial cell membranes, leading to the

release of intracellular content and the consequent cell death (Borges et al., 2013).

Additionally, gallic acid has been specifically identified as the compound responsible for

the growth inhibition of E. coli and S. enterica (Jayaprakasha et al., 2003).

Catechins and Epicatechin

Catechins are a class of phenolic compounds that are also abundant in the plant

kingdom. In the inhibition of bacterial growth, catechins function by inducing oxidative

stress and changing the membrane permeability of bacterial cells. A study showed that

catechins inhibit the growth of E. coli and S. enterica, much like gallic acid (Ma et al.,

2019). Epicatechin, another phenolic compound, functions in a similar way. Although

less research has been done on the antibacterial activity of epicatechin, one study found

that epicatechin displayed an inhibitory effect on Heliobacter pylori, a gram-negative

bacterium (Escandon et al., 2016). Because gallic acid, catechin, and epicatechin are

responsible for the inhibition of several gram-negative bacteria, grape seed extract’s

ability to inhibit gram-negative bacteria, a task which proves difficult for synthetic

antibiotics, likely comes from these compounds.

Action Mechanisms of Antibiotics

Beta-lactams

Beta-lactams are the most commonly used class of antibiotics. They function by

interfering with bacterial cell wall formation. Beta-lactams bind to specific enzymes

within the cell wall that are involved in the cross-linking of peptidoglycan, thus

preventing the synthesis of the peptidoglycan layer of bacterial cell walls. This

compromises the structural integrity of the cell wall, which leads to cell death (Bush &

Bradford, 2016).

Tetracyclines

Tetracyclines penetrate the cell walls of bacteria through passive diffusion. Once

inside the bacterium, tetracyclines inhibit protein synthesis in the bacterial ribosomes.

Because this action only inhibits the growth of bacterial cells, tetracyclines are only

effective against actively multiplying bacterial cells (Chopra & Roberts, 2001).

Fluoroquinolones

Fluoroquinolones are broad-spectrum antibiotics that work by inhibiting the DNA

replication and transcription processes of bacteria. They inhibit DNA gyrase and

topoisomerase IV—enzymes necessary for these processes. In doing so, fluoroquinolones

cause bacterial cell death (Patton & Reeves, 1988).

Aminoglycosides

Aminoglycosides are a class of broad-spectrum antibiotics that act by inhibiting

protein synthesis. Once inside the bacterial cell, they bind to the ribosomal RNA within

the bacterial ribosomes. This leads to an error in the synthesis of proteins, resulting in
harmful proteins being released into the cell, ultimately causing cell death (Krause et al.,

2016).

Macrolides

Macrolides target the peptide exit tunnel of the bacterial ribosome in order to

inhibit protein synthesis. These antibiotics prevent proteins from leaving the ribosome,

thus inhibiting the growth of bacterial cells. Like tetracyclines, macrolides are

bacteriostatic, meaning they are only effective against actively multiplying cells

(Vazquez-Laslop & Mankin, 2018).

Sulfonamides

Sulfonamides are a class of antibiotics that function by targeting dihydropteroate

synthase, an enzyme in the folic acid pathway. They aim to bind and inhibit this enzyme,

which is necessary for the synthesis of folate. Inhibiting the synthesis of folate in

bacterial cells leads to cell death. However, sulfonamides are not commonly used today

due to widespread resistance to them (Skold, 2000).

Conclusion

There have been many studies on the antibacterial activity of grape seed extract as

well as the natural compounds that are responsible for the inhibitory effects of grape seed

extract, but none have compared the antibacterial activity of grape seed extract to that of

synthetic antibiotics. The information communicated previously will be used to

determine the viability of grape seed extract as an antibiotic treatment.

 Chapter 3: METHODOLOGY

Overview

The viability of grape seed extract as an antibiotic treatment was determined by

comparing its bacterial inhibition properties to those of several antibiotics by using the

Kirby-Bauer disc diffusion test. The experiment was run in triplicate in order to validate

the observed results.

Apparatus and Materials

The grape seed extract used in this experiment was acquired from an online bulk

supplement supplier. This specific supplier was chosen because it offered a pure,

powdered form of grape seed extract.

Six antibiotics—penicillin, tetracycline, ciprofloxacin, tobramycin, polymyxin B,

and sulfamethoxazole/trimethoprin—were used in comparison to the grape seed extract.

These antibiotics were chosen because they are in assorted antibiotic classes and

represent different methods of bacterial growth inhibition. Penicillin acts by interfering

with bacterial cell wall formation; tetracycline and tobramycin act by inhibiting protein

synthesis; ciprofloxacin acts by inhibiting DNA synthesis; sulfamethoxazole/

trimethoprim acts by inhibiting the metabolic functions of bacteria; and polymyxin B acts

on bacterial cells by disrupting the cell membrane and neutralizing lipopolysaccharide.

The antibiotic discs were obtained from the Middle Tennessee State University science

department.

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus

subtilis were the bacteria used in this study. These species were chosen because they
display varying levels of antibiotic resistance. Samples of each bacterium were also

acquired from the MTSU science department.

Mueller-Hinton agar was chosen as the growth medium for this study. This agar is

standard for in-vitro antibiotic testing because it is a nonselective, nondifferential

medium, meaning that almost any type of bacteria can grow in it. Additionally, this type

of agar does not interfere with antibiotics, and its loose structure allows for better

diffusion of the antibiotics which allows for a more accurate zone of inhibition (Festus &

Emmanuella, 2020). The agar plates were acquired from the MTSU science department.

Procedures

Disc Preparation

To prepare the grape seed extract discs, 100mL of 70% isopropanol was placed

into a 250mL Erlenmeyer flask. To this, 1.5g of grape seed extract powder was added and

the mixture was brought to a boil. The heat was then reduced and allowed to simmer until

the isopropanol got below 20mL. The extraction was allowed to cool to room temperature

and then passed through a coffee filter. Once filtered, the solution was heated again until

the final volume was approximately 5mL. To saturate the solution onto ¼ inch sterile

discs, 0.2mL of the solution was placed onto each sterile disc. Fifteen discs were made.

Concurrently, 70% isopropanol was placed on sterile discs as well. Once these discs were

saturated, they were then allowed to evaporate off the residual isopropanol present. Once

all discs were dry, the process was repeated until there was nothing left of the grape seed

extract solution.
Experimental Procedure

Twenty-four plates were prepared with Mueller-Hinton agar, labeled with the

name of their assigned bacterium, and numbered from one to six. A sterile swab was used

to collect bacteria from the vials and streak the entire surface of the plate, forming a

confluent lawn. Six plates were used for each bacteria sample. After each plate had been

inoculated, a pair of forceps were used to place four disks on each plate. Ciprofloxacin,

tetracycline, polymyxin B, and tobramycin were placed on agar plates one, three, and five

while penicillin, sulfamethoxazole/trimethoprim, grape seed extract, and the isopropanol

control were placed on agar plates two, four, and six. The plates were then incubated at

35.5°C for 48 hours.

Data Analysis

After two days, the plates were removed from the incubator, and the zones of

inhibition were measured using a ruler. The results were recorded on a piece of paper,

and the mean zone of inhibition was calculated for each antibiotic and recorded in a table.

The data obtained from this experiment was analyzed quantitatively, and the zones of

inhibition were compared to determine if grape seed extract could realistically be used as

an antibiotic treatment.
 Chapter 4: FINDINGS AND DISCUSSION

Findings

Data Collected

Grape seed extract, isopropyl alcohol, and each antibiotic were tested against

Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa

in triplicate before their zones of inhibition, measured in millimeters, were averaged and

recorded below in Table 1.

Table 1, Average Zone of Inhibition Measurements for Antibacterial Agents

Average Zone of Inhibition (mm)

Bacteria
Antibacterial
Staphylococcus Bacillus Escherichia Pseudomonas
Agent
aureus subtilis coli aeruginosa
Grape Seed 13.7 0 0 0
Extract
Isopropyl Alcohol 0 0 0 0
(control)
Ciprofloxacin 29.7 26.3 48.7 32

Penicillin 20.3 0 0 0

Polymyxin B 0 15 15 19

Sulfamethoxazole/ 39.3 27 25.7 0

trimethoprim
Tetracycline 35.7 14 23.7 0

Tobramycin 26.7 20.3 18 26

Grape seed extract had an average zone of inhibition of 13.7mm in S. aureus and

0mm in B. subtilis, E. coli, and P. aeruginosa. As a negative control, isopropyl alcohol

had an average zone of inhibition of 0mm for S. aureus, B. subtilis, E. coli, and P.
aeruginosa. Conversely, Ciprofloxacin had an average zone of inhibition of 29.7mm in S.

aureus, 26.3mm in B. subtilis, 48.7mm in E. coli, and 32mm in P. aeruginosa. Penicillin

had an average zone of inhibition of 20.3mm in S. aureus and 0mm in B. subtilis, E. coli,

and P. aeruginosa. Polymyxin B had an average zone of inhibition of 0mm in S. aureus,

15mm in B. subtilis and E. coli, and 19mm in P. aeruginosa. Sulfamethoxazole/

trimethoprim had an average zone of inhibition of 39.3mm in S. aureus, 27mm in B.

subtilis, 25.7 in E. coli, and 0mm in P. aeruginosa. Tetracycline had an average zone of

inhibition of 35.7mm in S. aureus, 14mm in B. subtilis, 23.7mm in E. coli, and 0mm in

P. aeruginosa. Finally, tobramycin had an average zone of inhibition of 26.7mm in S.

aureus, 20.3mm in B. subtilis, 18mm in E. coli, and 26mm in P. aeruginosa.

Analysis of Data

Table 2, Reference Chart to Determine Susceptibility to Select Antibiotics (Adapted from BBL™ Sensi Disc
™ Antimicrobial Test Discs, July 2011)

Diameter of Zone of Inhibition (mm)

Antibiotic
Resistant Intermediate Susceptible

Ciprofloxacin ≤ 15 16 – 20 ≥ 21

Penicillin ≤ 28 — ≥ 29

Polymyxin B ≤8 9 – 11 ≥ 12

Sulfamethoxazole/ ≤ 10 11 – 15 ≥ 16
trimethoprim
Tetracycline ≤ 12 13 – 17 ≥ 18

Tobramycin ≤ 12 13 – 14 ≥ 15

While more extensive testing is required to determine the statistical significance

of the information, the data received from the research yielded interesting results.
 Figure 1, Staphylococcus aureus Plates

Staphylococcus aureus samples, as seen in Figure 1, saw varying levels of inhibition

from each of the antibacterial agents. In general, the tested antibiotics had larger zone of

inhibition measurements in these plates, which can be attributed to the gram-positive

structure of S. aureus. In accordance with Table 2, S. aureus was susceptible to

ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline, and tobramycin, and grape

seed extract saw moderate to high levels of inhibition. Conversely, S. aureus was

resistant to penicillin and polymyxin B.

 Figure 2, Bacillus subtilis Plates

As seen in Figure 2, gram-positive Bacillus subtilis samples also saw varying

levels of inhibition from each of the antibacterial agents. B. subtilis was susceptible to

ciprofloxacin, polymyxin B, sulfamethoxazole/trimethoprim, and tobramycin, but the

bacterium was resistant to penicillin and had an intermediate reaction with tetracycline.

Curiously, grape seed extract’s zone of inhibition was equal to the diameter of the paper

disc that it was on, meaning it had no significant effect on B. subtilis.

 Figure 3, Escherichia coli Plates

Escherichia coli, as seen in the plated samples in Figure 3, saw varying zone of

inhibition measurements, with ciprofloxacin having a significantly greater inhibitory

effect than the other antibacterial agents. Despite being a gram-negative bacterium, E.

coli was susceptible to a greater number of antibiotics than the other bacteria in this

study. According to Table 2, E. coli was susceptible to ciprofloxacin, polymyxin B,

sulfamethoxazole/trimethoprim, tetracycline, and tobramycin. However, E. coli was

resistant to penicillin, and grape seed extract displayed no inhibitory effect against the

bacterium.
 Figure 4, Pseudomonas aeruginosa Plates

Pseudomonas aeruginosa had the highest antibiotic resistance out of the selected

bacteria samples, as seen in Figure 4. In fact, grape seed extract had no effect on P.

aeruginosa. Of the tested antibiotics, neither penicillin, sulfamethoxazole/trimethoprim,

nor tetracycline had any effect on P. aeruginosa. Because the antibiotics’ zones of

inhibition were equal to the diameter of the paper disc they were on, P. aeruginosa was

completely resistant to the antibiotics. On the other hand, P. aeruginosa was susceptible

to ciprofloxacin, polymyxin B, and tobramycin.

Ciprofloxacin, penicillin, polymyxin B, sulfamethoxazole/trimethoprim,

tetracycline, and tobramycin were used in the study as a base of reference, as seen in

Table 2. Overall, ciprofloxacin and tobramycin were the most effective antibiotics as

every bacteria sample was susceptible to their inhibitory effects. In contrast, each bacteria

sample was resistant to penicillin, making penicillin the least effective antibiotic in the

study. Moreover, polymyxin B was effective against B. subtilis, E. coli and P.

aeruginosa with its zone of inhibition measurements lying in the susceptible range for

each bacterium. However, the zone of inhibition measurements for polymyxin B

remained in the resistant range for S. aureus. Sulfamethoxazole/trimethoprim proved to

be effective against S. aureus, B. subtilis, and E. coli, remaining well within its

susceptibility range. On the other hand, P. aeruginosa was resistant to the growth

inhibition of sulfamethoxazole/ trimethoprim. Finally, tetracycline remained within the

susceptibility range of S. aureus and E. coli, but it was only moderately effective against

B. subtilis with its zone of inhibition measurements lying within the intermediate range.

Tetracycline also proved ineffective against P. aeruginosa with its zone of inhibition

measurements lying within the resistant range.

Table 3, Average Zone of Inhibition Measurements for Antibacterial Agents

60

50

40
Average Zone of Inhibition Diameter (mm)

30

20

10

0
Pseudomonas aeruginosa
Escherichia coli
Staphylococcus aureus

Bacillus subtilis

Antibacterial Agent
Bacteria
Grape Seed Extract Isopropyl Alcohol (control) Ciprofloxacin Penicillin

Polymyxin B Sulfamethoxazole/trimethoprim Tetracycline Tobramycin

 Grape seed extract saw contrasting levels of inhibition on each of the bacteria

samples. From the results seen in Table 3, grape seed extract was only effective against

S. aureus. Not only was it completely ineffective against B. subtilis, E. coli, and P.

aeruginosa, but its zone of inhibition measurements for S. aureus were smaller than those

of every other tested antibacterial agent. Despite this, because S. aureus was resistant to

penicillin and polymyxin B, grape seed extract was more effective than those two

antibiotics. In fact, grape seed extract’s average zone of inhibition was above polymyxin

B’s susceptibility rating of 12mm. Grape seed extract did not reach the susceptibility

ratings of any other antibiotics, but it reached the intermediate ratings of

sulfamethoxazole/trimethoprim, tetracycline, and tobramycin.

Discussion

How does the in-vitro bacterial growth inhibition of grape seed extract compare to

those of penicillin, tetracycline, ciprofloxacin, tobramycin, sulfamethoxazole/

trimethoprim, and polymyxin B when tested against Staphylococcus aureus, Escherichia

coli, Pseudomonas aeruginosa, and Bacillus subtilis? In previous studies, grape seed

extract has shown strong antibacterial activity in both gram-positive and gram-negative

bacteria samples. In contrast, because of widespread antibiotic resistance, antibiotics have

varied in their antibacterial activity between gram-positive and gram-negative samples.

Because of this, the expected result of the study was that penicillin, tetracycline,

ciprofloxacin, tobramycin, sulfamethoxazole/trimethoprim, and polymyxin B would be

more effective in gram-positive S. aureus and B. subtilis, but grape seed extract would be

more effective in gram-negative E. coli and P. aeruginosa. The study rejects this
hypothesis as grape seed extract was only effective in S. aureus, a gram-positive

bacterium.

These results contrast previous studies in which grape seed extract showed

moderate zones of inhibition in a variety of bacterial samples including B. subtilis, P.

aeruginosa, and E. coli. This disparity may come as a result of the difference in solvents

used in the grape seed extract solutions in previous studies compared to this study.

Isopropyl alcohol was used as the solvent in this experiment, but previous studies used

either dimethyl sulfoxide, methanol, ethanol, or water. In an experiment that used

dimethyl sulfoxide, grape seed extract was most effective against E. coli whereas an

experiment that used methanol, ethanol, and water found that E. coli was least susceptible

to grape seed extract. In this study, grape seed extract was completely ineffective in

inhibiting the growth of E. coli. These drastic differences in results show that the solvent

used in the grape seed extract solution may have a significant effect on the outcome of

the experiment.

Another reason for the significant difference in results may have been the grape

seed extract used. The grape seed extract used in this experiment was obtained from a

different supplier than other studies, so it is possible that the extract could have been

processed differently, resulting in different phenolic contents and concentrations. Gallic

acid has been identified as a compound responsible for the growth inhibition of E. coli, so

it was possible that the specific grape seed extract used in this experiment had a lower

concentration of gallic acid. Catechins, a class of phenolic compounds, have also been

identified as inhibitors of E. coli growth. Because the grape seed extract used in this
experiment had no inhibitory effect on E. coli, it was likely that the extract had a lower

concentration of phenolic compounds such as gallic acid and catechins.

Although grape seed extract only proved to be effective in inhibiting the growth

of S. aureus, these results were still significant. S. aureus was more susceptible to grape

seed extract than penicillin and polymyxin B. Additionally, when tested against S.

aureus, grape seed extract performed intermediately compared to sulfamethoxazole/

trimethoprim, tetracycline, and tobramycin. Because the inhibitory effects of grape seed

extract were comparable to those of several antibiotics, this study determined that grape

seed extract could be a viable alternative treatment for Staphylococcus aureus infections.
 Chapter 5: CONCLUSION

Study Summary

This purpose of this study was to compare the bacterial growth inhibition of grape

seed extract to those of penicillin, tetracycline, ciprofloxacin, tobramycin,

sulfamethoxazole/trimethoprim, and polymyxin B when tested against Staphylococcus

aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis to determine the

viability of grape seed extract as an alternative antibiotic treatment. In order to test grape

seed extract, a solution was made using isopropyl alcohol. The Kirby Bauer disc

diffusion method was used to determine the susceptibility of the bacteria to the chosen

antibacterial agents.

Ultimately, grape seed extract only inhibited the growth of S. aureus, and it had

zero inhibitory effect on E. coli, P. aeruginosa, and B. subtilis. However, grape seed

extract proved to be more effective in inhibiting the growth of S. aureus than penicillin

and polymyxin B as the bacterium was resistant to both of these antibiotics. Grape seed

extract also performed intermediately when compared to sulfamethoxazole/

trimethoprim, tetracycline, and tobramycin. Grape seed extract’s results were promising,

and further research into its abilities could make it an effective alternative antibacterial

treatment in the future.

Limitations

One of the study’s main limitations is that it was performed in vitro instead of in

vivo. The researcher did not have the resources to conduct an in-vivo experiment, so the

study was performed in a controlled environment that could not account for the

complexity of the human body and its biochemical processes. It is impossible to know if
grape seed extract would behave the same way in a human subject without testing it in a

living organism. The purpose of the study was to determine if grape seed extract was a

viable antibiotic treatment, and it was deemed as one for Staphylococcus aureus

infections through in-vitro experimentation; however, further in-vivo testing must be

done to assess the safety of grape seed extract as an antibacterial treatment in humans

before it could truly be a viable treatment option.

Another limitation of the study was a constraint on time. The agar plates required

a 48-hour incubation period, so additional trials would have taken several days to weeks.

Because the researcher had a limited research window, it was not possible to do extensive

testing. Also, constant access to the MTSU research lab was not possible. With more time

for experimentation, additional testing could have been done to confirm the validity of

the results.

Implications

This study offers a variety of implications for the treatment of S. aureus

infections. Grape seed extract’s success in this experiment implies that it could eventually

be applied in vivo and used to treat S. aureus infections. These results can also be applied

to future research to determine the most efficacious way to administer grape seed extract

to those infected by S. aureus. As bacteria become more resistant to antibiotics, scientists

will look for other viable options to treat victims of bacterial infections, and this study

presents grape seed extract as one of those options. In fact, grape seed extract could

potentially be used to treat antibiotic-resistant strains of S. aureus such as MRSA.

Because it is resistant to the beta-lactam class of antibiotics, MRSA is very difficult to

treat; however, grape seed extract’s success against S. aureus in this study underscores it

as a potential treatment for this deadly bacterial strain.

Suggestions for Future Study

To address this research question to a further extent, experiments could be done

that compare the inhibition properties of grape seed extract to a wider variety of

antibiotics. If grape seed extract proved to be effective against an additional array of

antibiotics, its position as a viable alternative treatment for bacterial infections would be

further solidified. Another suggestion for additional research would be to test grape seed

extract against more bacteria to determine if it is effective against species other than S.

aureus. As discussed in Chapter 4, the solvent used in the grape seed extract solution

might play an important part in the bacterial inhibition of grape seed extract, so further

research could be done to determine if certain solvents enhance the antibacterial

properties of grape seed extract. A final suggestion for future studies would be to test

grape seed extract in vivo to determine if it performs similarly and has the same bacterial

inhibition properties in living organisms.

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