an ethnobotanical approach to finding antimicrobial
TRANSCRIPT
Pepperdine University Pepperdine University
Pepperdine Digital Commons Pepperdine Digital Commons
Featured Research Undergraduate Student Research
Summer 2013
An Ethnobotanical approach to finding antimicrobial compounds An Ethnobotanical approach to finding antimicrobial compounds
in wooly blue curls (Trichostema lanatum) using a Kirby-Bauer in wooly blue curls (Trichostema lanatum) using a Kirby-Bauer
disc diffusion assay disc diffusion assay
Matthew C. Fleming Pepperdine University
P. Matthew Joyner Pepperdine University
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Recommended Citation Recommended Citation Fleming, Matthew C. and Joyner, P. Matthew, "An Ethnobotanical approach to finding antimicrobial compounds in wooly blue curls (Trichostema lanatum) using a Kirby-Bauer disc diffusion assay" (2013). Pepperdine University, Featured Research. Paper 61. https://digitalcommons.pepperdine.edu/sturesearch/61
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Materials and Methods Plant collec0on • The plant specimens were found in the Santa Monica Mtns. near Pepperdine University . • We collected flowers, branches, and leaves of the plant sample.
Prepara0on of plant extracts • The plant specimen was soaked in MeOH for12-‐14hrs. (3x) • The solvent was then evaporated and the extract was re-‐dissolved in DMSO to its respec0ve concentra0ons.
Kirby-‐Bauer Disc Diffusion Assay (DDA) • Bacterial cultures were diluted to their respec0ve concentra0ons and a coPon swab was used to inoculate an agar plate. • A Sensi Disc Dispenser was used to introduce filter paper discs to the agar plates and 10μL of extract were used to inoculate each disc. • The plates were placed in a 37°C incubator for 18hrs. And the diameter of the zones of inhibi0on were measured in cm with a ruler. (C. xerosis was grown for 40hrs. due to a slow growth
rate.)
Results • The bar graphs show the diameter of the zone of inhibi0on for each concentra0on of extract used along with a posi0ve control (ampicillin) and a nega0ve control (DMSO). The error bars show the standard devia0on for each measurement.
An Ethnobotanical approach to finding an0microbial compounds in wooly blue curls (Trichostema lanatum) using a Kirby-‐Bauer disc diffusion assay
MaPhew C. Fleming and P. MaPhew Joyner Department of Biology, Natural Science Division, Pepperdine University, Malibu, California, USA
Introduc0on This study of medicinal plants incorporates the strategy of
ethnobotany, which tries to characterize why cultures use plants the way they do. Wooly blue curls is documented as being a medicinal plant for the Chumash people[1] and was shown to have an0bacterial proper0es from previous research from our lab. In order to study WBC’s an0bacterial proper0es, a panel of bacteria commonly found in separate anatomical habitats is u0lized. Differing bacteria with unique characteris0cs can specialize in separate niches[3]. Therefore, by using bacteria from different niches (in this study the skin niche and gut niche) we can bePer observe WBC’s an0microbial ac0vity.
Acknowledgements This research was funded by the Na0onal
Science Founda0on, Research Experience for Undergraduates, REU-‐Site Grant, #DBI-‐1062721 and the Natural Science Division of Pepperdine. We would also like to thank Victoria Hester for her help with plant collec0on and extract prepara0on.
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Abstract Plants can be an important source of crea0vity and
produc0on of new drugs. In this study, extracts of wooly blue curls (Trichostema lanatum) were made using DMSO and tested for an0microbial ac0vity on a panel of bacteria commonly found in separate ecological niches. Wooly blue curls (WBC) was chosen due to its being recorded as a strong disinfectant by the Chumash people. It was found that WBC does exhibit an0microbial ac0vity against gram posi0ve bacteria and not against gram nega0ve bacteria. However, gram nega0ve bacteria with reduced drug efflux func0on became suscep0ble to the WBC extract.
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SURBSURBSummer UndergraduateSummer Undergraduate
Research in BiologyResearch in Biology
Discussion The resistance of Gram nega0ve bacteria
may be explained due to efflux pumps, that are able to extrude an0microbials, and a second outer membrane that is hard for an0microbials to penetrate. Most plant pathogens are Gram nega0ve[1] , poten0ally because they have greater resistance to plant an0microbials. Gram posi0ve bacteria also have efflux pumps[1], however they lack the outer membrane causing them to be more suscep0ble. Therefore the presence or absence of specific efflux pumps may explain the varia0on of responses between Gram posi0ve bacteria. Due to ac0vity against a variety of bacteria and the role of efflux pumps in resistance, the an0microbial in WBC may have a conserved internal target among bacteria like the faPy acid cycle or a bacterial ribosome.
Figure 1: Collec0on of Trichostema lanatum
Figure 2: Example of a Kirby-‐Bauer disc diffusion assay. This shows the E. coli ΔtolC knockout mutant that allows for inhibi0on of the WBC extract.
Bacteria Niche Gram(+/-‐) 125mg/ml WBC inhibi.on (cm)
Staphylococcus epidermidis Skin + 1.11 + .07
Staphylococcus aureus Skin + .92 + .04
Corynebacterium xerosis Skin + 1.96 + .19
Enterococcus faecalis Gut + 1.16 + .02
Bacillus sub<lis Gut + 1.14 + .07
Bacillus megaterium Env. + 1.09 + .07
Salmonella typhimurium Gut -‐ 0 + 0
Escherichia coli WT Gut -‐ 0 + 0 Escherichia coli ΔtolC Gut -‐ 1.33 + .03
Table 1: Summary of DDA data.
References 1. Tegos, G., Stermitz, F. R., Lomovskaya, O., and Lewis, K. An<microbial Agents and Chemotherapy 46, no. 10 (2002): 3133–3141. doi:10.1128/AAC.46.10.3133-‐3141.2002, 2. Timbrook, J. (2007): 3. The Human Microbiome Project Consor0um. Nature 486, no. 7402 (2012): 207–214. doi:10.1038/nature11234, 4. Paul Cox and Michael J. Balick. Scien<fic American 270, no. 6 (1994): 82–87. 5. Davis, W. W. and Stout, T. R. Applied Microbiology 22, no. 4 (1971): 666–670. 125mg/ml 125mg/ml 125mg/ml
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Ampicillin Ampicillin Ampicillin
Ampicillin Ampicillin Ampicillin
Gram nega0ve E. coli ΔtolC with inhibited efflux pumps becomes suscep0ble to WBC extracts.
Gram posi0ve bacteria are suscep0ble to the WBC extract
WBC extracts did not inhibit wild type Gram nega0ve bacteria.
Figure 3: Comparison of the reac0ons of each bacteria with 125mg/ml WBC extract.
NI NT NT NI NI NI NI
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Graphs Legend: NI= No Inhibi0on NT= Not Tested
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Skin bacteria have a variety of responses.
Gut bacteria have a variety of responses.
This shows there is a broad range of reac0ons, from no zone of inhibi0on to almost a 2 cm zone of inhibi0on.
Further Study We would like to frac0onate the WBC
extract to determine the ac0ve component and test how effec0ve it is. Then we would like to test the an0microbial along with a known efflux inhibitor to see if there is a synergis0c effect between the two compounds. Another area of study that could prove beneficial is screening other Chumash medicinal plants to search for specimens that produce efflux inhibitors along with an0microbials.
Conclusions • Wooly blue curl extract does produce compounds that have an0microbial proper0es. • Wooly blue curl extracts do not affect all bacteria the same.
No inhibi0on