Transcript V. ovatum

An In Vitro Study of the Antimicrobial Activity of Vaccinium ovatum
Karina Chelnokova and Elena Anuryeva
Biology Department, Skyline College, San Bruno CA
Abstract
Methods
Results
As 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 extract’s 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.
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 5°C.
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 35°C 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 35°C for 48-96 hr.
Antimicrobial Peptide Assay
• Heated (56°C 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.
•
Hypothesis
An aqueous leaf extract of Vaccinium ovatum will inhibit gram-positive
bacteria. Antimicrobial peptides will be responsible for inhibition of
bacteria.
Background
•
•
•
•
•
.
0
Acetone
Leaf in
CFU/mL
100000
Figure 2. Inhibition of S. aureus by 500 mg/mL leaf extracts. Extracts
in polar solvents were most effective against the bacteria. Solvents alone
did not inhibit growth.
• The extracts from leaves and berries of V. ovatum inhibited grampositive 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
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1000000
5
Figure 1. Vaccinium
ovatum. California
Huckleberry, is an
Ericaceae that is native
to the Pacific Coast of
North America.
Discussion & Conclusion
Literature Cited
10
Water
14
22
45
*Leaf = freshly made
extract.
15
Ethanol
8
17
27
M. phlei
• Further studies could include investigation into the mechanism of
inhibition and isolation and purification of the active compounds.
20
Streptomycin
S. mutans
Figure 3. Well
diffusion assay
against M. pheli. The
aqueous leaf extract
was 31% as effective
as rifampin antibiotic.
25
Control
500 mg/mL Aqueous
Leaf Extract
Aqueous Berry
Antibiotic
• Aqueous leaf extract may treat S. aureus infections.
30
Zone of inhibition (mm)
• 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.
•
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).
Table 2. Well diffusion assay for S. mutans and M. phlei
Zone of inhibition (mm)
10000
Acknowledgements
Table 1. MIC of aqueous berry and leaf extracts (mg/mL).
Bacteria
Leaf Extract
Berry Extract
S. mutans
500
350
M. phlei
200
200
1000
Unheated extract
Heated extract
Control
Figure 4. Antimicrobial peptide assay. Heating did not affect
antistaphylococcal activity of the extract.
We thank Dr. Christine Case, Stephen Fredericks, Patricia Carter ,
Skyline’s 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.