Date of Award

8-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Advisor

Tzeng, Tzuen-Rong

Committee Member

Riley , Melissa B

Committee Member

Jiang , Xiuping

Committee Member

Gangemi , Joseph

Abstract

Multi-Drug Resistance (MDR) efflux pumps have been increasingly reported in Gram negative and Gram positive bacteria. These efflux mechanisms pump out a wide variety of structurally unrelated antimicrobials thus leading to a low susceptibility due to lowered intracellular concentrations. The activity of such antimicrobials can be restored by the inhibition of the multi drug efflux pumps. Several MDR pump inhibitors which inhibit the efflux mechanisms in bacteria have been identified. Reserpine and verapamil are two such inhibitors, showing considerable effect on the MDR pumps. But the concentrations required to achieve these effects are too high to be clinically relevant. Reserpine has adverse effects such as neurotoxicity. The identification and development of safe and effective inhibitors of bacterial efflux pumps is needed. Crude leaf extracts of goldenseal (Hydrastis canadensis) have been shown to have such effects.
This dissertation researches the MDR pump inhibitory activity of goldenseal leaf extract and the separation of its constituents. First, three forms of goldenseal samples supplied by the Sleepy Hollow Farm, GA were evaluated for their antimicrobial properties. Minimum inhibitory concentration (MIC) of all different forms of goldenseal i.e, powders, liquid extracts, and retention solids, in five different ratios of root/leaf combinations was determined against MDR bacterial strains of Staphylococcus aureus and Campylobacter jejuni. The antimicrobial activity of goldenseal samples was also determined against human intestinal beneficial bacteria, Lactobacillus acidophilus. Goldenseal liquid extracts were found to have the highest activity against the MDR bacteria while showing minimum impacts on the L. acidophilus.
Second, goldenseal leaf extract was assayed for its MDR efflux pump inhibitory activity against MDR pumps belonging to different pump superfamilies in both S. aureus and C. jejuni. Goldenseal increased the potency of different antimicrobials against the MDR bacteria when combined in sub lethal doses (≤ 0.5MIC) by at least 2-folds decrease in MICs of antimicrobials against S. aureus strains and 16-fold in C. jejuni strain. Quantitative real time RT-PCR and ethidium bromide uptake/efflux studies indicated that goldenseal leaf extract represses the genes encoding these MDR pumps in both the bacteria.
Finally, five active bands exhibiting potential MDR efflux pump inhibitory activity were identified in thin layer chromatography and bioautographic studies. The identity of these bands could not be established by GC/MS which might be due to the volatility of the compound. LC/MS analysis led to a list of possible compounds in these active bands. In conclusion, the results of this work indicate that one or more constituents in goldenseal leaf extract exhibit MDR efflux pump inhibitory activity.

Included in

Microbiology Commons

Share

COinS