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Phytochemicals increase the antibacterial activity of antibiotics by acting on a drug efflux pump.

Ohene-Agyei T, Mowla R, Rahman T, Venter H - Microbiologyopen (2014)

Bottom Line: In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe-arg-β-naphthylamide (PAβN).Subsequently, promising products have been tested for their ability to inhibit efflux.We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug-resistant strains and which might be potentially useful lead compounds for an EPI discovery program.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, United Kingdom.

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Related in: MedlinePlus

Accumulation of fluorescent compounds in the Escherichia coli strains used in this study. E. coli BW25113 (black line) and BW25113 ΔAcrB (broken line) cells were grown to an OD660 of 0.5. Cells were harvested, washed three times in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 5 mmol/L MgSO4 and resuspended to a final OD660 of 0.5. (A–C). Cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 μmol/L) for complete deenergization. (D–F). Cells were incubated in the presence of glucose (20 mmol/L) to generate metabolic energy before the fluorescence trace was started. Ethidium (2 μmol/L), Hoechst 33342 (0.125 μmol/L), or TMA-DPH (0.25 μmol/L) were added at the points indicated by the arrows and the increase in fluorescence was followed over time.
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fig01: Accumulation of fluorescent compounds in the Escherichia coli strains used in this study. E. coli BW25113 (black line) and BW25113 ΔAcrB (broken line) cells were grown to an OD660 of 0.5. Cells were harvested, washed three times in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 5 mmol/L MgSO4 and resuspended to a final OD660 of 0.5. (A–C). Cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 μmol/L) for complete deenergization. (D–F). Cells were incubated in the presence of glucose (20 mmol/L) to generate metabolic energy before the fluorescence trace was started. Ethidium (2 μmol/L), Hoechst 33342 (0.125 μmol/L), or TMA-DPH (0.25 μmol/L) were added at the points indicated by the arrows and the increase in fluorescence was followed over time.

Mentions: Ethidium, Hoechst 33342 and TMA-DPH all share the property that they are virtually nonfluorescent in aqueous solution while undergoing a large increase in fluorescent quantum yield when in a hydrophobic environment such as when in a lipid bilayer or intercalated into DNA. Hence, the passive permeation of these compounds into bacterial cells can be followed by the increase in their fluorescence. It is clear from Figure 1A–C that the fluorescence (and hence passive permeation) of all three fluorescent compounds are identical in E. coli cells which have been deenergized by the addition of the protonophore CCCP. Therefore, there is no intrinsic difference in permeability between the cells used in this study. However, when the cells are energized by the addition of glucose, there is a marked decrease in fluorescence between the wild type (resistant) and ΔAcrB cells (Fig. 1D–F). This decrease in fluorescence is representative of AcrB-mediated efflux of the fluorescent compounds. Combined, the results mean that any effects on the MICs for the test compounds observed would be due to the active AcrB-mediated efflux of the compounds and not due to intrinsic differences in the membrane permeability between the strains used in the study.


Phytochemicals increase the antibacterial activity of antibiotics by acting on a drug efflux pump.

Ohene-Agyei T, Mowla R, Rahman T, Venter H - Microbiologyopen (2014)

Accumulation of fluorescent compounds in the Escherichia coli strains used in this study. E. coli BW25113 (black line) and BW25113 ΔAcrB (broken line) cells were grown to an OD660 of 0.5. Cells were harvested, washed three times in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 5 mmol/L MgSO4 and resuspended to a final OD660 of 0.5. (A–C). Cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 μmol/L) for complete deenergization. (D–F). Cells were incubated in the presence of glucose (20 mmol/L) to generate metabolic energy before the fluorescence trace was started. Ethidium (2 μmol/L), Hoechst 33342 (0.125 μmol/L), or TMA-DPH (0.25 μmol/L) were added at the points indicated by the arrows and the increase in fluorescence was followed over time.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263512&req=5

fig01: Accumulation of fluorescent compounds in the Escherichia coli strains used in this study. E. coli BW25113 (black line) and BW25113 ΔAcrB (broken line) cells were grown to an OD660 of 0.5. Cells were harvested, washed three times in 50 mmol/L potassium phosphate buffer (pH 7.0) containing 5 mmol/L MgSO4 and resuspended to a final OD660 of 0.5. (A–C). Cells were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (10 μmol/L) for complete deenergization. (D–F). Cells were incubated in the presence of glucose (20 mmol/L) to generate metabolic energy before the fluorescence trace was started. Ethidium (2 μmol/L), Hoechst 33342 (0.125 μmol/L), or TMA-DPH (0.25 μmol/L) were added at the points indicated by the arrows and the increase in fluorescence was followed over time.
Mentions: Ethidium, Hoechst 33342 and TMA-DPH all share the property that they are virtually nonfluorescent in aqueous solution while undergoing a large increase in fluorescent quantum yield when in a hydrophobic environment such as when in a lipid bilayer or intercalated into DNA. Hence, the passive permeation of these compounds into bacterial cells can be followed by the increase in their fluorescence. It is clear from Figure 1A–C that the fluorescence (and hence passive permeation) of all three fluorescent compounds are identical in E. coli cells which have been deenergized by the addition of the protonophore CCCP. Therefore, there is no intrinsic difference in permeability between the cells used in this study. However, when the cells are energized by the addition of glucose, there is a marked decrease in fluorescence between the wild type (resistant) and ΔAcrB cells (Fig. 1D–F). This decrease in fluorescence is representative of AcrB-mediated efflux of the fluorescent compounds. Combined, the results mean that any effects on the MICs for the test compounds observed would be due to the active AcrB-mediated efflux of the compounds and not due to intrinsic differences in the membrane permeability between the strains used in the study.

Bottom Line: In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe-arg-β-naphthylamide (PAβN).Subsequently, promising products have been tested for their ability to inhibit efflux.We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug-resistant strains and which might be potentially useful lead compounds for an EPI discovery program.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, United Kingdom.

Show MeSH
Related in: MedlinePlus