Mohammed Abbas & Martin Cordero Biology Department, Skyline College, San Bruno CA Evaluating the Antibacterial Properties of Ceanothus thyrsiflorus Mohammed Abbas & Martin Cordero Biology Department, Skyline College, San Bruno CA Acknowledgements First and foremost, we would like to thank our mentor Dr. Christine Case for donating her time, energy, and intellect to ensure a first-class learning environment, providing us and our peers with invaluable laboratory experience and biological knowledge. Patricia Carter and Ruth Arce, our lab technicians, for their help and countless hours spent in lab readily available to us, and Stephen Fredricks for handling the logistics between Skyline College and SACNAS. Lastly, we thank SACNAS for giving Skyline College the opportunity to participate in a national scientific conference. Literature Cited Borchardt, J. R., et al ”Antioxidant and Antimicrobial Activity of Seed from Plants of the Mississippi River Basin.” Journal of Medicinal Plants Research 3(10): Hedges, Ken Santa Ysabel Ethnobotany. San Diego Museum of Man Ethnic Technology Notes, page 15. Mitscher, Lester A “Coevolution: Mankind and Microbes.“ The American Chemical Society and American Society of Pharmacognosy 71(3): Moerman, D. (ed). “Native American Ethnobotany.” University of Michigan Salazar-Aranda, R., et al “Antimicrobial and Antioxidant Activities of Plants from Northeast of Mexico.“ Evidence-based Complementary and Alternative Medicine (epub doi /). Background There are many other types of antibiotics, each with unique mechanisms that vary in their approach. However, due to the rapid reproduction of bacteria and overuse and misuse of antibiotics, antibiotic resistant pathogens have emerged as a major health concern (Mistcher). New antimicrobics must be found. Plants have historically been used as remedies for a variety of diseases. Ceanothus spp. have been used by Native Americans to treat wounds and gastrointestinal illnesses (Moerman, Hedges). C. coeruleus flower extracts inhibit S. aureus (Salazar-Aranda),and C. americanus seed extracts have been proven to be effective free radical scavengers (Borchardt). Abstract Antibiotics have been widely used to treat bacterial infections since the 1940’s. However their over-use has caused selection of antibiotic resistant pathogens. Indigenous North American people traditionally used Ceanothus thyrsiflorus to treat urinary, intestinal, and respiratory infections. Our purpose was to determine whether C. thyrsiflorus has antibacterial activity. Methanolic leaf extracts (500 mg/mL) were prepared. Well-diffusion assays were performed to test the extract against three gram-positive (Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) bacteria and three gram-negative (Escherichia coli, Salmonella enterica, Shigella sonnei). The methanolic Ceanothus extract inhibited S. aureus, with a 3-mm zone of inhibition. C. thyrsiflorus did not inhibit the gram-negative bacteria. Serial dilutions were used to determine the minimal inhibitory concentration (125 mg/mL) and the minimal bactericidal concentration (250 mg/mL). There is no lysozyme activity in the extract. The antibacterial fraction was unable to be isolated by Sephadex gel filtration. Further investigation of the active compound is warranted for confirmation. Materials & Methods Extract Preparation C. thyrsiflorus (Figure 1) was collected from Skyline College. Leaves were ground in 95% methanol (500 mg/mL) with a mortar and pestle and centrifuged at 78  g for two minutes. The supernatant was tested for antibacterial activity. Well-diffusion Assay 4-mm holes were aseptically punched into nutrient agar plates using a sterile cork borer. The plates were inoculated with: Escherichia coli (ATCC 11775) Salmonella enterica (ATCC 14028) Shigella sonnei (ATCC 9290) Staphylococcus aureus (ATCC 27659) Streptococcus pyogenes (ATCC 12228) Enterococcus faecalis (ATCC 19433). 20 µL methanolic Ceanothus leaf extract (500 mg/mL) was placed into each well; 20 µL 95% methanol was pipetted into the control well. The cultures were incubated at 35 ° C for 24 hours. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) Determination Serial dilutions of methanolic Ceanothus leaf extracts were made in a well cell plate. Each well was inoculated with 200 µL S. aureus. The plate was incubated for 24 hours at 35°C. After incubation, the plates were observed for bacterial growth. Wells with no growth were subcultured on a nutrient agar plate to determine the MBC. Lysozyme Activity A 1:10 dilution of leaf extract (500 mg/mL) was made in lysozyme buffer and inoculated with Micrococcus luteus in a spectrophotometer tube. The absorbance was recorded at 30-second intervals for five minutes. This was repeated replacing leaf extract with egg-white lysozyme (control). Gel Filtration A 500 mg/mL extract was filtered through a Sephadex (G-25) column. 25-drop aliquots were collected across 20 test tubes. 200 µL of each collection were used in a well-diffusion assay against S. aureus. The extracts were also centrifuged at 313  g, 704  g, and 1252  g and gel filtrated in separate trials. Each collected filtered extract was tested for inhibition as well. Results The methanolic Ceanothus leaf extract (500 mg/mL) did not inhibit any gram-negative bacteria. Of the three gram-positive bacteria being tested, only S. aureus was inhibited by the extract (Figures 2 & 3). The MIC was 125 mg/mL, and the MBC was 250 mg/mL. The extract had no lysozyme activity (Figure 4). The active compound was not separated by gel filtration. Gel filtration: No zones of inhibition were obtained. Discussion & Conclusion C. thyrsiflorus leaf extract was tested against three gram-positive bacteria and three gram-negative bacteria. The methanolic leaf extract inhibited S. aureus but did not inhibit S. pyogenes or E. faecalis. C. thyrsiflorus is a possible source of an antistaphylococcal agent, however further research is required for confirmation. Paper chromatography with an appropriate solvent or high performance liquid chromatography may isolate the active compound. Figure 2. The methanolic Ceanothus extract is 30% as effective as penicillin against S. aureus. Figure 3. Methanolic Ceanothus extract (500 mg/mL) inhibited gram-positive S. aureus. Error bars: 1 S. D. Figure 1. Ceanothus thyrsiflorus, a plant native to the Pacific Coast, is in the Rhamnaceae. Aim To evaluate the antimicrobial properties of Ceanothus thyrsiflorus. Figure 4. No lysozyme activity was found in the methanolic Ceanothus extract.