ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 1

EFFECTS OF ANTIOXIDANTS ON STREPTOMYCIN RESISTANT Escherichia coli

Karsen Hise, Logan Schroeder, and Reilly Kuhn

Loras College, Department of Biology

1450 Alta Vista Street
Dubuque, Iowa 52001

ABSTRACT
Millions of lives have been saved since the introduction of antibiotics to society, but as a
result of the escalation of resistant bacteria over the last decade, antibiotics are becoming less
effective in medical treatment. This new threat risks the lives of millions of people and requires
new research efforts to quickly manage a potential epidemic. Thus, the goal of this experiment
was to determine the extent to which antioxidant containing compounds, extracts of kale, ginger,
and grape seed, inhibited the growth of a streptomycin resistant strain of Escherichia coli (E.
Coli). We tested the inhibitory effects of these antioxidant compounds by measuring their zones
of inhibition using the Kirby-Bauer disc diffusion method. These results were compared to
ampicillin, streptomycin, and water controls. Ampicillin was the only compound which resulted
in a measurable zone of inhibition. Statistical analysis showed that the mean zone of inhibition
of ampicillin was significantly different from the streptomycin and water controls. Ampicillin
was also significantly different from kale, ginger, and grape seed extracts, which displayed no
zones of inhibition. Overall, the results did not support our prediction that these specific
antioxidant compounds would inhibit the growth of streptomycin resistant E. coli; therefore,
further experimentation is required to determine the antimicrobial effects of antioxidant
containing compounds.
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 2

INTRODUCTION

Each year, at least 2 million people contract antibiotic resistant bacterial infections due to
the overuse and misuse of antibiotics, allowing these resistant bacteria to spread into the
microbiome (CDC, 2018a). These bacterial infections, which were once easily treated, become
resistant to medication and are extremely difficult to kill. There are around 23,000 deaths each
year due to the limitations that current antibiotics have (CDC, 2018b). For example, Klebsiella
pneumoniae, commonly known as pneumonia, has developed an insensitivity to ampicillin,
making it more difficult to treat (Exner et al., 2017). One of the reasons for this increase in
resistance is due to the lack of development of new antibiotics; because of the limited amount of
time that each antibiotic can be used, in conjunction with the high cost of production and low
cost of sale, many pharmaceutical companies are no longer developing new antibiotics (Ventola,
2015).
One of the dangers associated with the use of antibiotics is the creation of Reactive
Oxygen Species (ROS). ROS are unstable, oxygen-containing molecules that take electrons from
surrounding molecules to increase their own stability (Yarosz & Chang, 2018). ROS are essential
for many lifesaving mechanisms. When immune cells phagocytose bacteria, they create ROS that
help kill the invaders (Salganic, 2001). If these radicals are not controlled, they continue
gathering electrons, become more reactive, and then can damage proteins, lipids, DNA, and other
essential biomolecules (Doughari et al., 2009). Once formed, these free radicals grow very
quickly. Antioxidants help counter the effects of ROS by reacting with the free radicals to inhibit
their growth, thus providing protection for the body’s essential biomolecules (Lobo et al., 2010).
Furthermore, in some cases the use of antibiotics against harmful bacteria, such as ciprofloxacin,
has induced the formation of ROS (Goswami et al., 2011). As a result, Goswami et al. (2011)
suggests that the combined effect of antibiotics and antioxidants has a powerful effect in fighting
harmful bacterial advancement in the body.
Halvorsen et al. (2002) examines antioxidant contents in various fruits, vegetables, and
roots. They suggest that three of the most antioxidant rich foods in the human diet are ginger,
kale, and grape seed. The antioxidant concentration of the three foods are 3.76, 2.34, and 1.45
mmol/100g, respectively, yielding a high antioxidant concentration in comparison to other
antioxidant containing foods (Halvorsen et al., 2002). Malviya et al. (2014) shows the
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 3

effectiveness of concentrated antioxidant pomegranate peels against many strains of bacteria

including Salmonella and Staphylococcus, yielding excellent antibacterial activities in relation to
their free radical scavenging abilities. In addition, Hu et al. (2014) examines the effects of leafy
green vegetables and their antimicrobial effects on gram-positive and gram-negative bacteria.
These vegetables have greater antimicrobial effects on gram-negative bacteria in comparison to
gram-positive bacteria. Similarly, all vegetables have greater antimicrobial effects in comparison
to a controlled water extract (Hu et al., 2004). Although kale, ginger, and grape seeds are known
to have a high concentration of antioxidant components, they have not been examined in their
effectiveness against harmful bacteria. We, therefore, proposed to analyze the effectiveness of
these substances on Escherichia coli (E.coli), a gram negative bacteria.
Ventola (2015) reports that, in general, gram-negative bacteria are more likely than gram-
positive bacteria to develop antibiotic resistance. In particular, gram-negative bacteria are
becoming resistant to nearly all antibiotic options on the market (Ventola, 2015). Given that
resistance is rapidly acquired and there are very few antibiotic options for treatment against some
strains of gram-negative bacteria, it seems essential to find other options to combat the harmful
effects of this bacteria on antibiotic resistance and overall human health. Streptomycin, however,
is a common antibiotic that is effective in conjunction with gram-negative bacteria, such as E.
coli. Due to the ease of access of both E. coli and streptomycin, we made E. coli bacteria that
was resistant to streptomycin to simulate the effects of antibiotic resistant bacteria. Therefore, we
proposed an alternative treatment consisting of the use of antioxidants found in ginger, kale, and
grape seeds which are suspected to be effective in fighting against the spread of streptomycin
resistant gram-negative bacteria. In order to compare the antimicrobial effects of these
antioxidants, they were tested against ampicillin, another antibiotic that is effective against gram-
negative bacteria. Therefore, we predicted that the antioxidants in kale, ginger, and grape seeds
will counteract the effects of streptomycin resistance, inhibiting the growth of the E. coli.

METHODS

Agar and Streptomycin Plates

LB (Lysogeny broth) agar was made by mixing 18.4 g of LB agar powder in 800 mL of
distilled water at a final concentration of 15 g/L agar, 10 g/L tryptone, 5 g/L yeast extract, and 5
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 4

g/L sodium chloride (NaCl) (Sigma-Aldrich). The solution was autoclaved for 20 minutes. A 10
mg/mL solution of streptomycin was prepared by adding 100 mg of streptomycin (Sigma-
Aldrich) to 10 mL of distilled water. After the agar solution was allowed to cool to approximately
55 ℃, 800 µL of the 10 mg/mL streptomycin solution was added and then mixed using a stir bar.
The final concentration of streptomycin in the LB agar plates was 10 µg/mL. The agar was
poured into twenty-four 95 mm petri dishes and allowed to sit overnight at room temperature.

Lysogeny Broth with Streptomycin

Two solutions of LB were made by dissolving 1.4 g of LB powder in 70 mL of distilled
water at a final concentration of 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl (Sigma-
Aldrich). The broth was autoclaved and allowed to cool to room temperature. In one of the
solutions, 70 µL of the 10 mg/mL streptomycin solution (made as described above) was added to
the broth for a final concentration of 10 µg/mL streptomycin. Following broth preparation, 5 mL
of LB ± streptomycin was aliquoted into 28 sterile 15 mL conical tubes.

Inducing Antibiotic resistance in E.coli

Streptomycin resistance was induced in E. coli using the protocol put forth by the
Department of Biology at Loras College (Shealer et al., 2018). E.coli was obtained from an agar
plate culture using a sterile inoculating loop and then added to a sterile tube of 5 mL LB without
streptomycin. The broth was placed in a shaking 37.0 ℃ incubator at 200 rpm for 24 hours.
Following this incubation, 0.5 mL of the E. coli broth culture was added sterilely to each of the
12 tubes containing 5 mL of 10 µg/mL streptomycin LB. The tubes were incubated in a shaking
37.0 ℃ incubator at 200 rpm for 48 hours. Following the 48 hour incubation, 0.1 mL of the
E.coli culture was transferred to an agar plate containing streptomycin and incubated for an
additional 48 hours at 37.0 ℃ (Shealer et al., 2018). Small colonies of streptomycin resistant E.
coli were evident and these colonies were collected and re-suspended in LB broth with
streptomycin and incubated for 24 hours. The resulting culture was again aliquoted onto LB agar
with streptomycin petri dishes using the same technique as above.

Kirby-Bauer Disk Diffusion

 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 5

Filter paper discs were sterilized for 15 minutes using an autoclave. Extracts of ginger
(Ginger People, 1 tbsp), kale (Dynamic Health, 1 tbsp), and grape seed (Nature’s Plus,
25mg/1mL) were also purchased. Following sterilization,
10 µL of grape seed extract was added to 4 separate paper
discs and allowed to dry. The same procedure was
completed for kale, ginger, and distilled water. Ampicillin
(10 µg) and streptomycin (10 µg) antibiotic susceptibility
test discs were obtained from Becton, Dickinson and
Company. Then 0.25 mL of the streptomycin resistant E.
coli was added to each of the LB with streptomycin agar
petri dishes. The infused discs were added to the center of
each of the plates: grape seed, ginger, kale, distilled
water, ampicillin, and streptomycin (n = 4). The Kirby-
Bauer Disk Diffusion Test was used to analyze the zone
of inhibition of the streptomycin resistant bacteria as Fig. 1. Measurement of zones of inhibition on
ampicillin plate using caliper.
seen in Figure 1 (Hudzicki, 2009). The plates and lids
were inverted and stored at 37°C overnight. Following the incubation time, the zones of
inhibition were measured to the nearest millimeter using a caliper (Hudzicki, 2009).

Statistical Analysis
Significance between control and experimental groups was examined using ANOVA and
post-hoc tests. A p-value of <0.05 was regarded as significant.

RESULTS

Mean zone of inhibition differed significantly among the 6 groups (F5,17=18071.011,

p<0.001). The Tukey post-hoc test on mean zone of inhibition shows that the water, grape seed,
kale, ginger, and streptomycin groups were not significantly different from each other (Figure 2).
However, the ampicillin group is significantly different from the rest (p < 0.001).
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 6

Fig. 2. Mean zone of inhibition of E. Coli after exposure to 6 types of treatment disks and
overnight growth. The ampicillin disks caused the largest zone of inhibition and was
significantly different than all other treatment groups (mean = 27.27mm + 0.20, p <0.001). All
other treatment groups did not cause inhibited growth (mean = 0). Error bars are at 95% CI.

DISCUSSION

The results from the present study do not support our prediction that that the antioxidant
rich compounds could inhibit the growth of streptomycin resistant E. coli. Following the
methods of the Kirby-Bauer test, it was shown that ampicillin, another gram-negative antibiotic,
was the only successful compound in inhibiting growth of streptomycin resistant E. coli. The
mean zone of inhibition for the ampicillin was 27.27 + 0.20 mm. The zone of inhibition for the
streptomycin control was 0 mm, indicating that we were successful at creating a strain of
streptomycin resistant E. coli. However, the mean zones of inhibition for the three antioxidant
compounds were also 0 mm. These results indicate that they were not successful at inhibiting the
growth of bacteria, even at their maximum concentrations.
The kale extract did not have an inhibitory effect on the growth of streptomycin resistant
E. coli. However, Hu et al. (2004) found that Chinese kale had antimicrobial effects against two
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 7

species of Pseudomonas, a genus of gram-negative bacteria, but it should be noted that there was
no antibiotic resistance induced in Pseudomonas (Hu et al., 2004). Similarly, Gupta and
Ravishnakar (2005) found that commercial ginger paste displayed more antimicrobial activity
against E. coli, compared to carrot, garlic, and turmeric antioxidant extracts. The E. coli used in
Gupta and Ravishnakar’s (2005) study was also not subjected to the induction of antibiotic
resistance. Furthermore, Stankovic et al. (2008) utilized grape seed extract as a treatment against
ROS in human lymphocytes. They found that treatment of lymphocytes with grape seed extract,
specifically at a concentration of 2.5μg/mL, decreased the amount of ROS damage in the
lymphocytes. Thus, it is speculated that the kale, ginger, and grape seed extracts used in the
present study did not have an effect on E. coli because it was resistant to streptomycin prior to
exposure of these antioxidant compounds. However, it is still thought that these antioxidants do
have beneficial antimicrobial properties as shown by Hu et al. (2004), Gupta and Ravishnakar
(2005), and Stankovic et al. (2008). It is possible that our experimental groups did not display
zones of inhibition since they were whole vegetable extracts rather than protein extracts. Hu et al
(2004) used pure protein extracts, and their results did display antimicrobial activity in Chinese
kale, among other vegetables. We think that our extracts may not have been as concentrated as
the protein extracts used in Hu et al (2004), and thus were unable to display antimicrobial effects.
If the antioxidant compounds were successful at inhibiting the growth of streptomycin
resistant bacteria, we would have seen a similar zones of inhibition to that on the ampicillin
plates. With the high prevalence of antibiotic resistance, it is essential that there is continued
research on other methods of killing harmful bacteria. Antibiotics often induce enhanced levels
of ROS, and Goswami et al. (2012) suggests that the combined effect of antibiotics and
antioxidants will be productive in the treatment of harmful bacterial infections. While the
antioxidant compounds did not yield any zone of inhibition, it is important to continue
researching the viability of antioxidant compounds and the effects they have on inhibiting
bacterial growth. Though our study looked at all the compounds independently, future research
might test the effect of antibiotics in conjunction with the experimental antioxidant compounds
on the same disc. Specifically, combining ampicillin with kale, ginger, or grape seed extracts
could be used to see if there is a stronger inhibitory effect as Goswami et al. (2012) suggests.
Thus, if it is possible for antioxidant food compounds to immediately destroy ROS that are
created by the antibiotic, the compounds could work together successfully.
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 8

ACKNOWLEDGEMENTS

In addition to those listed in the literature cited, we would like to thank Dr. Shealer and
Mary Jo Bowman for their assistance throughout the experiment, particularly with helping us
determine which procedures to perform, deciding which data analysis tests to run, and finally for
helpful criticism of the content of this paper. Specifically, we are thankful for the Loras College
Science Department for providing the materials needed throughout this experiment. This work
was supported in part from funds supplied by Loras College.
 ANTIOXIDANTS AND ANTIBIOTIC RESISTANCE 9

LITERATURE CITED

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Books:

Shealer, D., Sinha, A.,Czarnecki, D., (2018). Principles of Biology 116 Laboratory
Manual. Loras College Biology Department (38-42)

Websites:

CDC. (2018a, May 29). The Development of Antibiotic Resistance Bacteria. Retrieved
September 19, 2018, from https://www.cdc.gov/antibiotic-
use/community/about/antibiotic-resistance-faqs.html
CDC. (2018b, September 10). The Biggest Antibiotic-Resistant Threats in the U.S.
Retrieved September 19, 2018, from
https://www.cdc.gov/drugresistance/biggest_threats.html