This document summarizes research on the antimicrobial properties of blueberries against harmful bacteria. It finds that blueberry juice was highly effective at inhibiting the growth of Staphylococcus aureus but had little effect on Proteus vulgaris and Enterobacter aerogenes. The antimicrobial activity of blueberries is attributed to their high levels of phenolic compounds. While blueberries showed promise against S. aureus, more research is needed to determine if they can effectively treat infections from other antibiotic-resistant bacteria.

1. 12/02/14
Antimicrobial Properties of Blueberries on Harmful Bacteria
By: Jill Engeli
Introduction
Different kinds of berries have been tested to see how effective they are at killing
bacteria. This is important because if berries have strong antimicrobial properties they can be
used to treat bacterial infections instead of using antibiotics. We have become reliable on
antibiotics to kill bacteria, and as a result, bacteria have evolved to become antibiotic-resistant.
In the last several years, the frequency and spectrum of antimicrobial-resistant infections have
increased in both the hospital and the community (Cohen, 1992). The antimicrobial properties of
berries have also been studied to see how effective they are at killing foodborne pathogens. Due
to this problem, scientists must look to other resources and strategies to combat these bacteria
and pathogens.
Blueberries are known for being high in antioxidants. Blueberries have the highest
antioxidant capabilities of all fruits and vegetables…[and are recognized] as a super food (Mack,
2011). They have many health benefits as they are low in calories and high in fiber, but the most
important benefit of blueberries is that they have antimicrobial properties. Their antimicrobial
properties are due to the various phenolics and organic acids that are present in blueberries (Qui
and Wu, 2014). Phenolics are aromatic compounds that are derived from phenols. A relationship
between bacterial growth and the amount of phenolic compounds has been discovered. The stage
of growth to the production of phenolic compounds…showed to be powerful antioxidants… (Al-
Ani, Al-Haliem, Trawfik, 2010). The more phenolic compounds that are present, the less
bacterial growth that is present. Recent research found that phenolic compounds from blueberries
can inhibit cancer cell proliferation (Wu, 2014). These compounds are the key to understanding
how to use blueberries to create antimicrobial drugs to help battle the antimicrobial-resistant
bacteria.
One of the major foodborne pathogens is Staphylococcus aureus. It is a Gram-positive,
coccus shaped bacteria that is commonly found in the respiratory tract and on the skin. About
25% of humans and animals are carriers of this bacteria (Anonymous, 2006). Only carriers can
transmit this bacteria to other people which can cause a problem in the food industry. The most
common way for food to be contaminated with S. aureus is through contact with food workers
who carry the bacteria or through contaminated milk and cheeses (Anonymous, 2006). Once the
bacteria comes into contact with food, it quickly multiplies and produces toxins which can cause
food poisoning if the contaminated food is digested. The problem with S. aureus food poisoning
is that it cannot be treated with antibiotics so a different solution is needed to deal with the
serious cases of food poisoning.
Proteus vulgaris is a Gram-negative, rod-shaped bacteria that is commonly found in the
intestinal tracts, soil and water. This bacteria is well-known for causing urinary tract infections.

2. To treat urinary tract infections antibiotics are used, but ingestion of cranberry juice could also
help clear up the infection. Since cranberry juice and blueberry juice are similar in the amount of
phenolic compounds present, blueberry juice could possibly be used to help treat urinary tract
infections. Another issue that occurs with P. vulgaris is that it commonly causes nosocomial
infections. Hospitals and healthcare facilities are breeding grounds for many kinds of bacteria.
This is a problem because the bacteria can grow, evolve and possibly become resistant to
antibiotics. The antimicrobial properties of blueberries could be used to help prevent the
evolution of P. vulgaris into an antibiotic-resistant bacteria.
Blueberries could also be effectively used to prevent Enterobacter aerogenes growth in
hospitals. E. aerogenes is a Gram-negative, rod-shaped bacterium that can easily become
resistant to antibiotics. Since this bacteria can easily become antibiotic-resistant, different
antibiotics must be administered to prevent the onset of sepsis. It has also been known to cause
an increase in histamine production in foods which sometimes leads to an outbreak in food
poisoning (Taylor, et. al, 1978). Food poisoning occurs from histamines when high
concentrations of histamines in certain foods are ingested. The high concentrations of histamines
causes histamines to be released from mast cells which can cause rashes, diarrhea, stomach
cramps and many other symptoms. The use of blueberries and their antimicrobial properties
could be extremely beneficial in combating E. aerogenes in hospitals and in preventing
histamine food poisoning.
The objective of this research was to see how efficient and effective blueberries were on
stopping the growth of harmful bacteria. Previous research had been conducted using
cranberries, acai berries, strawberries, raspberries and blueberries on S. aureus and positive
results were observed (Poh, et. al, 2012). These researchers found that cranberry juice was very
effective at controlling S. aureus growth with blueberry juice being the next effective.
Cranberries and blueberries have strong antimicrobial properties due to the amount of phenolic
compounds present in them. Blueberries also have a high antioxidant capacity which may allow
them to have possible protective effects against human degenerative diseases (Wu, 2014). Using
this information and research, it can be deduced that blueberries could have a positive effect on
controlling the bacterial growth of foodborne pathogens.
Methods
Materials:
 1 mL pipettes
 100 mL beaker
 3 Dilution bottles
 70% ethyl alcohol solution
 Ampicillin, Gentamicin and Streptomycin antibiotic discs
 Blueberries
 Bunsen burner
 Cheesecloth
 Distilled water

3.  Enterobacter aerogenes culture
 Incubator
 Proteus vulgaris culture
 Staphylococcus aureus culture
 Sterilized knife and tweezers
 Sterilized Petri Plates
 Tap water
Naturipe organic blueberries were purchased from Wal-Mart in Platteville, WI. The
blueberries were washed with tap water, sterilized with 70% ethyl alcohol solution and
then rinsed with distilled water. After sterilization, they were cut with a sterilized knife
and the contents of the blueberries were put into a sterile beaker. These contents were
then poured over a beaker that was covered with cheesecloth to filter out the seeds and
other tissues of the blueberries. Dilution bottles were opened aseptically and 99 mL of
distilled water was poured into each bottle while 1 mL of blueberry juice was poured into
the first bottle. Serial dilutions were carried out for the pour plate quantitative plating
method (See Figure 1). The remaining blueberry juice and the dilution bottles were stored
at 4°C.
The study was conducted in the bacteriology laboratory in Boebel Hall at the
University of Wisconsin-Platteville. Three different types of bacteria were used in this
study: Staphylococcus aureus, Proteus vulgaris and Enterobacter aerogenes. Nutrient
agar was used for P. vulgaris and E. aerogenes while nutrient agar with yeast was used
for S. aureus. To test the activity of the bacteria, the Kirby-Bauer disc method was used.
Prepared antibiotic doses in milligrams of ampicillin (10 μg), gentamicin (10 μg) and
streptomycin (10 μg) were used for the control to see the susceptibility of the bacteria.
The remaining plates were plated with the bacteria and the serial dilutions. All the plates
were incubated at 37°C for a week and then the zone of inhibition was measured and
recorded in millimeters. For each test there were three replicates.
Data and Results
Staphylococcus aureus
Antimicrobial activity was seen in all of the plates except for the 10-6 dilution
(Table 2). S. aureus was susceptible to gentamicin and streptomycin as its zone of inhibition was
larger than 15 mm respectively; it was also resistant to ampicillin as its zone of inhibition was
less than 22 mm (Table 1). The most significant zone of inhibition occurred at the 101 dilution
(38.5 mm). No zone of inhibition was seen at the 10-6 dilution.
Proteus vulgaris
Antimicrobial activity was only seen in two of the dilutions (10-5, 10-7) (Table 2).
The 10-5 dilution had a zone of inhibition of 26.5 mm while the 10-7 had a zone of inhibition of
10 mm. P. vulgaris was susceptible to gentamicin and streptomycin; it was also resistant to
ampicillin as the zone of the inhibition on the plate was less than 22 mm (Table 1).
Enterobacter aerogenes

4. Antimicrobial activity was seen in four of the dilutions (10-3, 10-5, 10-6, and 10-
7) (Table 2). E. aerogenes was susceptible to gentamicin and streptomycin; it was also resistant
to ampicillin as its zone of inhibition was less than 22 mm (Table 1). The most significant zone
of inhibitions occurred on the 10-6 and 10-7 dilution plates (57.5 mm, 46 mm).
Discussion
The most antimicrobial activity was observed in the S. aureus cultures while the least
activity was observed in the P. vulgaris cultures. All of the replicates of S. aureus had
significantly less growth on them which is what was expected. Blueberries are potent
antimicrobial agents because they contain a high number of phenolic compounds and flavonoids.
The mechanism of how these phenolic compounds work is still not understood, but they are
believed to be a major reason why blueberries are so effective at hindering bacterial growth of
foodborne pathogens.
The blueberry juice had a slight effect on the growth of E. aerogenes. The largest zone of
inhibitions occurred on the most diluted plates. The most diluted plates contained miniscule
amounts of blueberry juice. Since E. aerogenes is known for being able to quickly evolve, it is
possible that it was able to evolve and not be affected by the blueberry juice in the plates that
contained the least diluted juice. If being exposed to higher concentrations of blueberry juice did
not have an effect on E. aerogenes, then the use of blueberries would not be an efficient
treatment for someone who is dealing with an infection caused by this bacteria. There was not a
significant amount of evidence that blueberry juice was effective on stopping the bacterial
growth of E. aerogenes therefore the hypothesis cannot be supported.
Zone of inhibitions were only present on two plates that were inoculated with P.
vulgaris. Also, some of the plates did not have much bacterial growth on them. This could be due
to the fact that the bacterial culture was not completely healthy during the time the experiment
was done. Since the culture was possibly not healthy during the time of the experiment, this
could have skewed the data and given inaccurate results. If that is not the case, then the data
shows that blueberry juice was ineffective at stopping the growth of P. vulgaris. Since the
bacterial growth was not hindered, blueberry juice would not be an effective way to treat
infections caused by P. vulgaris.
Overall, blueberry juice seemed to only have a significant effect on S. aureus and no
significant effect on E. aerogenes and P. vulgaris. Possible reasons for the ineffectiveness of the
blueberry juice was that E. aerogenes could have evolved and protected itself from being
hindered by the higher concentrations of blueberry juice and that the P. vulgaris culture was not
completely healthy at the time of the experiment. The S. aureus data from this experiment
coincides with the S. aureus data from an experiment done by Al-Anin. In Al-Anin’s experiment,
different kinds of fruit juices were used to see if they hindered harmful bacterial growth. It was
found that S. aureus was greatly affected by all of the fruit juices, while P. vulgaris was only
affected by two out of the four fruit juices used. It is possible that the antimicrobial properties of
blueberries can only stop the growth of foodborne pathogens since S. aureus infections
commonly come from improper food handling. The other bacterial cultures used are known for
causing infections in hospitals and urinary tract infections which are not foodborne illnesses. Due

5. to the lack of significant data, the effectiveness of blueberry juice in hindering bacterial growth
cannot be supported. Further studies and more thorough research needs to be done to see if
blueberries could actually be used instead of antibiotics in treating antibiotic-resistant bacteria.
Since we rely on antibiotics so much in today’s society, another option needs to be found so we
can stop the ever increasing amount of antibiotic-resistant bacteria.

6. 1Tables & Figures
Figure 1: Serial Dilution and Pour Plate Quantitative Plating Method
Table 1: Interpretation of Zone Diameters of Test Cultures
Abbreviation Resistant (mm or
less)
Intermediate (mm
range)
Susceptible (mm or
more)
Ampicillin Am 20 21-28 29
Gentamicin Gm 12 13-14 15
Streptomycin S 11 12-14 15
Table 1 was used in determining if the bacterial cultures were either resistant, intermediate or susceptible to the
antibiotics used for the controls. If the bacteria were resistant, there was no zone of inhibition or a small zone of inhibition and
large growth of bacteria. None of the bacteria had an intermediate zone of inhibition. All bacteria were susceptible to gentamicin
and streptomycin so they had large zone of inhibitions and little to no growth of bacteria.
Table 2: Average zone of inhibition of different blueberry juice dilutions and antibiotic discs on
S. aureus, P. vulgaris and E. aerogenes.
Blueberry Juice Dilutions Antibiotic
Disc
101 10-1 10-2 10-3 10-4 10-5 10-6 10-7 Am Gm S
Types of Bacteria
Staphylococcus aureus 38.5 22.5 27.5 32 16.5 8 --** 12.5 -- 42 22
Proteus vulgaris -- -- -- -- -- 26.5 -- 10 -- 25 21
Enterobacter
aerogenes
-- -- -- 19.5 -- 25 57.5 46 -- 18 28
*Diameter of zoneof inhibition measured in millimeters (mm). Datarepresented as mean of three readings; **No zone
of inhibition (--); Disc diameter = 6mm; Am = 10 μg; Gm=10 μg; S=10 μg.

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