Karina Chelnokova and Elena Anuryeva Biology Department, Skyline College, San Bruno CA

1. Figure 3. Well diffusion assay against M. pheli. The aqueous leaf extract was 31% as effective as rifampin antibiotic. *Leaf = freshly made extract. Figure 1.Vaccinium ovatum. California Huckleberry, is an Ericaceaethat is native to the Pacific Coast of North America. Figure 2. Inhibition of S. aureusby 500 mg/mL leaf extracts. Extracts in polar solvents were most effective against the bacteria. Solvents alone did not inhibit growth. Figure 4. Antimicrobial peptide assay. Heating did not affect antistaphylococcal activity of the extract. 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 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 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. 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 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. • 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). • 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. aretraditionally 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 Huang, Y.,D. Nikolic,and S. Pendland. “Effects of cranberry extracts and ursolic acid derivatives on P-fimbriated Escherichia coli, COX-2 activity, pro-inflammatory cytokine release and the NF-κβ transcriptional response in vitro.” Pharmaceutical Biology 47(1): 18–25, 2009. Johnson, B. J., B. Lin,and R. A. Rubin. “Media acidification by Escherichia coli in the presence of cranberry juice.” BMC Research Notes 2: 226, 2009. Lee, J., C. E. Finn, and R. E. Wrolstad. “Comparison of Anthocyanin Pigment and Other Phenolic Compounds of Vaccinium membranaceum and Vaccinium ovatum Native to the Pacific Northwest of North America.” Journal of Agriculture and Food Chemistry 52 (23): 7039–44, 2004. Lee, Y. L.,I. N.Wadie,and J. Owens. “Anti-microbial Activity of Urine after Ingestion of Cranberry: A Pilot Study.” Evidence Based Complement Alternative Medicine 7(2): 227–232, 2010. Magarinos, H. L. E., C. Sahr, and S. D. C. Selaive. “In vitro Inhibitory Effect of Cranberry (Vaccinium macrocarpum Ait.) Juice on Pathogenic Microorganisms.” Applied Biochemistry and Microbiology 44(3):300–4, 2008. Miyako, Y., N. Khalef, and K. Matsuzaki. “Solubility Enhancement of Hydrophobic Compounds by Cosolvents: Role of Solute Hydrophobicity on the Solubilization Effect.”International Journal of Pharmaceutics 393 (1-2):48-54, 2010. Neto, C. "Cranberry and Its Phytochemicals: A Review of In Vitro Anticancer Studies." American Society for Nutrition 137:186-193, 2007. Turner, A., S. Chen,and D. Nikolic. “Coumaroyl Iridoids and a Depside from Cranberry (Vaccinium macrocarpon).” Journal of Natural Products 70(2): 253–8, 2007. . AcknowledgementsWe 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.