Scientific Poster

1

An In Vitro Study of the Antimicrobial
Activity of Vaccinium ovatum
Karina Chelnokova and Elena Anuryeva Biology
Department, Skyline College, San Bruno CA
Karina Chelnokova and Elena Anuryeva Biology
Department, Skyline College, San Bruno CA
Methods Extract Preparation Leaves and berries
of V. ovatum were collected in Pacifica,
California (Figure 1). Plant parts were ground
into a homogeneous substance using mortar and
pestle in the following solvents 95 ethanol,
acetone and water. Water was used to prepare
berry extract. Extracts had a final
concentration of 500 mg/mL. Extracts were
filtrated through the cheesecloth and
refrigerated at 5C. Well Diffusion
Assay Nutrient agar was inoculated with
Escherichia coli (ATCC 11775), Staphylococcus
aureus (ATCC 27659), Streptococcus pyogenes (ATCC
12228), Streptococcus mutans (ATCC 25 175), or
Mycobacterium phlei (Wards 85W1691). 6-mm wells
were made in the agar plates using a cork borer
and filled with 50 µL of an extract.
Antibiotic discs (Hardy Diagnostics) were used
as positive controls penicillin (10 µg/disk),
streptomycin (10 µg/disk), rifampin (5 µg/disk),
and bacitricin (10 µg/disk). Water, 95
ethanol, and acetone were used as negative
controls. Nutrient agar plates were incubated
at 35C for 48-120 hr. Minimum Inhibitory
Concentration (MIC) Both leaf and berry
aqueous extracts were serially diluted from 500
mg/mL to 150 mg/mL. The well diffusion assay
was used for the MIC assay. Nutrient agar was
inoculated with S. aureus, S. mutans, and M.
phlei. All the nutrient agar plates incubated
at 35C for 48-96 hr. Antimicrobial Peptide
Assay Heated (56C for 30 min), unheated
aqueous leaf extracts, and sterile water
(control) were added to S. aureus in nutrient
broth and incubated at 37?C for 1 hour. Plate
counts were used to determine the number of
surviving bacteria.

• Results
• V. ovatum extracts had no effect on
  gram-negative bacteria. They did inhibit
  gram-positive bacteria.
• Extracts in polar solvents (water and acetone)
  showed the highest level of inhibition of
  gram-positive bacteria. Solvents, alone, did not
  inhibit bacterial growth (Figure 2).
• The MIC against M. phlei is 200 mg/mL (Table 1).
• The antimicrobial activity of the berry extract
  was 51 less effective than the antibiotic
  rifampin against M. phlei (Figure 3).
• The leaf extract was 35 less effective than
  penicillin against S. aureus.
• For S. mutans the extract was 63 less effective
  than the antibiotic (bacitracin). The extracts
  had no antimicrobial activity against S. pyogenes
  (Table 2).
• There was no significant difference (10) between
  the antibacterial activity of heated and unheated
  leaf extracts. (Figure 4).

AbstractAs bacteria become increasingly
resistant to antibiotics, alternative
antimicrobials are needed to treat bacterial
infections. For centuries, plants have been used
by many cultures to treat a variety of
infections. One such plant, Vaccinium, has been
used by west coast Native Americans to treat a
variety of ailments. While much focus has been
placed on the antimicrobial activity of V.
macrocarpon, little is known about other species
of Vaccinium. The purpose of our study is to
investigate the antimicrobial activity of V.
ovatum. An aqueous leaf extract (0.5 g/mL)
inhibited growth of gram-positive bacteria,
Staphylococcus aureus, but not gram-negative
Escherichia coli bacteria in a well-diffusion
assay. The crude extract is 35 less effective
than a commercial penicillin disk (10 µg). The
minimum inhibitory concentration of this extract
against S. aureus was 0.3 g/mL. The
antimicrobial activity of the plant does not
appear to be caused by proteins because heat (56
C for 30 min) did not affect the extracts
activity. We are testing the aqueous extract
from leaves and berries of V. ovatum against
other gram-positive bacteria to ascertain its
optimum potential use.
Table 2. Well diffusion assay for S. mutans and M. phlei Table 2. Well diffusion assay for S. mutans and M. phlei Table 2. Well diffusion assay for S. mutans and M. phlei
500 mg/mL Aqueous Zone of inhibition (mm) Zone of inhibition (mm)
500 mg/mL Aqueous S. mutans M. phlei
Leaf Extract 8 14
Aqueous Berry 17 22
Antibiotic 27 45

• Discussion Conclusion
• The extracts from leaves and berries of V. ovatum
  inhibited gram-positive bacteria.
• The aqueous extract was most effective suggesting
  the antibacterial compound is nonpolar (6).
• Antibacterial action is not due to antimicrobial
  peptides.
• Aqueous leaf extract of V. ovatum may provide an
  alternative treatment for multi-drug resistant M.
  phlei.
• An aqueous berry extract of V. ovatum could be
  used in oral hygiene products
• Aqueous leaf extract may treat S. aureus
  infections.
• Further studies could include investigation into
  the mechanism of inhibition and isolation and
  purification of the active compounds.

HypothesisAn aqueous leaf extract of Vaccinium
ovatum will inhibit gram-positive bacteria.
Antimicrobial peptides will be responsible for
inhibition of bacteria.

• Background
• For centuries, plants have been used by many
  cultures to treat a variety of disease including
  bacterial infections.
• Plants produce a variety of antimicrobial
  phytochemicals (7).
• Native Americans used Vaccinium spp. to treat
  wounds. In many other cultures Vaccinium spp. are
  traditionally used in folk medicine for the
  management of diverse conditions including
  urinary and stomach problems (3, 5).
• Because of its long history in folk medicine,
  Vaccinium spp. supplements have entered the
  nutritional market (8).
• As bacteria become increasingly resistant to
  antibiotics, alternative antimicrobials are
  needed to treat infections.
• The antibacterial properties of V. microcarpum
  are known, however, little is known about other
  species of Vaccinium (3, 1).
• V. ovatum may contain the antibacterial
  properties needed to treat antibiotic-resistant
  bacteria.

• Literature Cited
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• Johnson, B. J., B. Lin, and R. A. Rubin. Media
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• Magarinos, H. L. E., C. Sahr, and S. D. C.
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  (Vaccinium macrocarpum Ait.) Juice on Pathogenic
  Microorganisms. Applied Biochemistry and
  Microbiology 44(3)3004, 2008.
• Miyako, Y., N. Khalef, and K. Matsuzaki.
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  Journal of Pharmaceutics 393 (1-2)48-54, 2010.
• Neto, C. "Cranberry and Its Phytochemicals A
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.
AcknowledgementsWe thank Dr. Christine Case,
Stephen Fredericks, Patricia Carter , Skylines
SACNAS Chapter and the MESA Center for making our
research possible and providing us with the
opportunity to attend SACNAS National Conference.
We also thank Carleton Cheng who assisted with
research and provided the original photographs of
the plant.
Table 1. MIC of aqueous berry and leaf extracts (mg/mL). Table 1. MIC of aqueous berry and leaf extracts (mg/mL). Table 1. MIC of aqueous berry and leaf extracts (mg/mL).
Bacteria Leaf Extract Berry Extract
S. mutans 500 350
M. phlei 200 200