Hamza Fakhri, Margaret Nguyen and Christine Case Biology Department, Skyline College, San Bruno CA. Hamza Fakhri, Margaret Nguyen and Christine Case Biology Department, Skyline College, San Bruno CA.
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Biology Department, Skyline College, San Bruno CA
Hamza Fakhri, Margaret Nguyen and Christine Case
Biology Department, Skyline College, San Bruno CA
Figure 4. E. coli growth curve. The control had a doubling time of 210 min. The doubling time was > 8 times slower in the plant extract. Trial 1 never reached the doubling time in the time span of the experiment (30 hr). Error bars = 1 S.D.
Figure 1.Cortaderia selloana, pampas grass, is a grass native to South America. It is grown around the world in regions with Mediterranean and temperate climates, including areas of Africa, Australia, Europe, New Zealand, and North America.
Figure 3. Well diffusion assay of ethyl acetate extracts. Ethyl acetate leaf and root extracts demonstrated the most inhibition zones against E. coli. L= leaf, R= root, S= seeds, F= flower, C= control.
Figure 2. Well diffusion assay. Ethyl acetate roots, flowers, leaves, and stems extracts inhibited all microorganisms tested (E. coli,St. aureus, and Sa. cerevisiae). Root and leaf extracts showed the greatest inhibition zones. Error bars = 1 S.D.
Investigation of Antimicrobial Activity of
Cortaderia selloana, Pampas Grass
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.
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 4°C. Extracts were filtered through Whatman no. 1 filter paper. The filtrates were then left to dry at 25°C. 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 37°C. 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 37°C, 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 37°C. 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 4°C 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 ether:1 acetone) was used to isolate the antibacterial compound(s) in the ethyl-acetateleaf 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 56°C for 30 min. Heated extract, unheated extract, or a control (corn oil) was inoculated with E. coli and incubated at 37°C for 24 to 48 hr. Heterotrophic plate counts were used to count surviving bacteria.
•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.
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).
Cortaderia selloana has antimicrobial properties that can inhibit growth of foodborne pathogens.
• 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.
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_poisoning/
3Okada, 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.
4Sanchez, 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.
5Scientific Committee on Food. 2002. Report on Risk Profile on the Microbiological Contamination of Fruits and Vegetables Eaten Raw. Belgium: European Commission.
6World Health Organization. 2007. “Food Safety and Foodborne Illnesses.” www.who.int/mediacentre/factsheets/fs237/en/
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 President’s Innovation Fund.