Scientific Poster

1
Investigation of Antimicrobial Activity of
Cortaderia selloana, Pampas Grass
Abstract Foodborne infections affect millions
of people in both developed and developing
countries. Many of these infections are due to
contaminated food or water. Therefore, there is a
constant need for new antimicrobial agents to
prevent survival and growth of bacteria in food.
The use of natural compounds from plants can
provide an alternative approach against foodborne
pathogens. Cortaderia selloana, pampas grass,
is an invasive species outside of its
native South American habitat. Our hypothesis is
that this plant has antimicrobial activity
against pathogens which contribute to its
success. The aim of our work is to explore the
antimicrobial properties of C. selloana. In this
experiment, extracts of roots, flowers, leaves,
and stems were made in ethyl acetate, water, 75
ethanol, or 95 methanol. The extracts were then
screened against Staphylococcus
aureus (gram-positive), Escherichia coli
(gram-negative) bacteria, and Saccharomyces
cerevisiae (fungus) using well diffusion assays.
The ethyl-acetate leaf extract (333
mg/ml) inhibited all of the test microbes with
zones of inhibition ranging from 13.1 to
14.0 mm. The minimum bactericidal concentration
of the leaf extract against Sa.
cerevisiae and against E. coli is 41.7 mg/mL. The
chemical nature of the antimicrobial
compound(s) was investigated. The effectiveness
of the leaf extract preventing E. coli survival
and growth in fresh produce was determined. The
results indicate that C. selloana has
natural antimicrobial properties to prevent
foodborne gram-negative bacterial infections.
Methods 1. Preparation of Plant Extracts
Cortaderia selloana specimens were collected from
Skyline College. Using a mortar and pestle,
roots, flowers, leaves, and stems were ground in
95 ethyl acetate, distilled water, 75 ethanol,
or 95 methanol then left overnight at 4C.
Extracts were filtered through Whatman no. 1
filter paper. The filtrates were then left to dry
at 25C. The dried extracts were resuspended in
10 mL of the solvent used for primary extraction
to a final concentration of 5000 mg/mL. 2. Well
Diffusion Assay Saccharomyces cerevisiae (ATCC
9763) fungus and Escherichia coli (ATCC 11775)
and Staphylococcus aureus (ATCC 27659) bacteria
were spread separately onto the surface of
nutrient agar (NA) plates. Wells were made in the
agar with a 6-mm cork borer and 100 µL of each
extract was deposited into each well. Solvents
used for each extract were employed as controls.
Plates were incubated for 24 hr at 37C.
Antimicrobial activity was detected by the
presence of an inhibition zone surrounding the
wells. 3. MIC/MBC Determinations Serial
dilutions of leaf and root extracts (5 mg/mL to
166 mg/mL) were made in cell well plates with
nutrient broth. Wells were inoculated with 5 µL
of E. coli or Sa. cerevisiae. After 24 hr
incubation at 37C, subcultures of wells with no
visible growth were made on NA plates. 4.
Determination of bacterial Growth Curve The
ethyl acetate leaf extract was evaporated then
reconstituted in edible cooking oil. The extract
was added to nutrient broth in Nephelo flasks to
a final concentration of 100 mg/mL. Another flask
containing nutrient broth served as the control.
All flasks were inoculated with 100 µL of 24-hr
E. coli culture. The flasks were incubated at
37C. Absorbance was recorded at 570 nm. 5.
Survival of E. coli on Salad Vegetables Lettuce
and spinach were purchased and cut into eight 4
cm by 4 cm pieces. The pieces were submerged in
100 mL inoculum with 1.8 108 CFU/mL E. coli in
a 1000-mL beaker. To increase the number of cells
attached, the eight pieces were kept at 4C for
24 hr in bags. Leaf extract was dried and
reconstituted in corn oil to a final
concentration of 5000 mg/mL. The lettuce and
spinach pieces were placed in new plastic bags
with 8 mL of 166 mg/mL leaf extract and gently
agitated on an orbital shaker for 5 min. Survival
of E. coli in treated and control pieces was
determined using heterotrophic plate counts. 6.
Chromatography Paper chromatography (in 9
petroleum ether1 acetone) was used to isolate
the antibacterial compound(s) in the
ethyl-acetate leaf extract. The dried
chromatogram was cut into 0.5-cm pieces, which
were used in an agar diffusion assay. 7.
Bacteriocin Determination Leaf extract
(ethyl-acetate extract reconstituted in corn oil
to a final concentration of 166 mg/mL) was heated
to 56C for 30 min. Heated extract, unheated
extract, or a control (corn oil) was inoculated
with E. coli and incubated at 37C for 24 to 48
hr. Heterotrophic plate counts were used to count
surviving bacteria.
Results Ethyl acetate extracts of all plant
parts inhibited all of the microorganisms tested
(E. coli, St. aureus, and Sa. cerevisiae) (Figure
2). 333.3 mg/mL ethyl acetate leaf and root
extracts were most affective against E. coli
(Figure 3). The ethyl acetate leaf and root
extracts had a lower MIC and MBC against E. coli
than against Sa. cerevisiae (Table 1). C.
selloana leaf extract (ethyl acetate extract
reconstituted in corn oil) decreased the growth
rate of E. coli at 100 mg/mL (Figure 4). C.
selloana leaf extract in corn oil did not affect
the survival of E. coli on lettuce and spinach.
The fractions of the ethyl acetate leaf extracts
containing antibacterial compounds were found at
Rf values of 0.55 and 0.71. Heating the leaf
extract in corn oil did not change its
antibacterial action. Hence, the antibacterial
compound is not a protein.
Table 1. Minimum Inhibitory (MIC) and Minimum Bactericidal (MBC) Concentrations of Ethyl Acetate Leaf and Root Extracts Against E. coli and Sa. cerevisiae. Table 1. Minimum Inhibitory (MIC) and Minimum Bactericidal (MBC) Concentrations of Ethyl Acetate Leaf and Root Extracts Against E. coli and Sa. cerevisiae. Table 1. Minimum Inhibitory (MIC) and Minimum Bactericidal (MBC) Concentrations of Ethyl Acetate Leaf and Root Extracts Against E. coli and Sa. cerevisiae.
  MIC (mg/mL) MBC (mg/mL)
Leaf E. coli 20.8 41.7
Root E. coli 20.8 41.7
Leaf Sa. cerevisiae 41.7 83.3
Root Sa. cerevisiae 41.7 83.3
Discussion Conclusion C. selloana extracts
inhibit fungi, gram-negative and gram-positive
bacteria. Fresh fruits, vegetables and beef
have the potential to be contaminated with E.
coli bacteria (2). This research shows that C.
selloana leaf extract inhibits E. coli growth and
survival. Treatment with the leaf extract used
as salad dressing could be an inexpensive natural
method for decreasing E. coli growth on
foodstuffs. The antimicrobial action most
likely is not due to a protein because heating
did not affect antibacterial activity. Future
experimentation will determine appropriate
methods for the nontoxic extraction of the active
compounds and explore the potential to be used as
salad dressing. Further testing of C. selloana
leaf extracts needs to be done to identify the
chemical composition of the bactericidal
compound(s).
Aim Cortaderia selloana has antimicrobial
properties that can inhibit growth of
foodborne pathogens.
Background Foodborne infections are estimated
to affect 76 million people in the U.S and cause
1.8 million deaths worldwide (6). Outbreaks
of illnesses caused by bacteria have been linked
to the consumption of a wide range of vegetables
(5). The resistance of microorganisms to
antibiotics is on the rise making treatment more
difficult (4). There is a need for inexpensive
food preservatives to improve food safety.
Plants have been historically used as
antimicrobial agents (4). Cortaderia selloana
(Poaceae family), is an invasive plant found
worldwide in temperate regions (Figure 1) (3).
There are only 17 fungal pathogens recorded
specifically for C. selloana. Of those recorded,
only one is pathogenic to the plant (1). The
purpose of this work is to investigate the
antimicrobial properties of C. selloana. We
examined whether ethyl acetate extracts of C.
selloana inhibit growth and survival of
Escherichia coli bacteria in food.
Literature Cited 1. Froude, V. A. 2002.
Biological Control Options for Invasive Weeds of
New Zealand Protected Areas. Science for
Conservation 199. 2 Foodborne Illnesses Common
Bacteria and Viruses that Cause Food Poisoning.
2005. www.foodborneillness.com/ecoli_food_poisonin
g/ 3 Okada, M., R. Ahmad, and M. Jasieniuk. 2007.
Microsatellite Variation Points to Local
Landscape Plantings as Sources of Invasive Pampas
Grass (Cortaderia selloana) in California.
Molecular Ecology 16(23) 4956-71. 4 Sanchez,
E., S. Garcia, and N. Heredia. 2010. Extracts of
Edible and Medicinal Plants Damage Membranes of
Vibrio cholerae." Applied and Environmental
Microbiology 76(20) 6888-94. 5 Scientific
Committee on Food. 2002. Report on Risk Profile
on the Microbiological Contamination of Fruits
and Vegetables Eaten Raw. Belgium European
Commission. 6 World Health Organization. 2007.
Food Safety and Foodborne Illnesses.
www.who.int/mediacentre/factsheets/fs237/en/
Acknowledgements Our fellow lab classmates who
share the same level of passion for scientific
inquiry. Pat Carter and Kylin Johnson for readily
providing us with all of the materials and
equipment needed to conduct our research. MESA
club for being a valuable resource in our
research. SACNAS for giving us the opportunity
for an enriching experience. Most importantly,
our mentor Dr. Christine Case who has
continuously supported us through her guidance
and inspiration. Our research was funded by the
Presidents Innovation Fund.