Evaluation of cell membrane integrity as a potential antimicrobial target for plant products

Abstract

Background: There is urgent need to discover new antimicrobial compounds with diverse chemical structures and mechanisms of action due to increasing new and re-emerging infectious diseases. Additionally, appearance of undesirable side effects of certain antibiotics and increasing resistance to antibiotics in current clinical use is also a cause for concern. Bacterial cell membranes are a possible target for developing new antibacterial drugs since membrane-based efflux pump systems play an important role in bacterial pathogenicity and antimicrobial resistance in bacteria. Hence, the objective of our study was to evaluate bacterial membrane integrity of two species of bacteria; Staphylococcus aureus and Pseudomonas aeruginosa, in the presence of ethanolic leaf extracts of two plant species Callistemon citrinus and Vernonia adoensis from Zimbabwe.

Methods: Bacterial efflux pump inhibition using both leaf extracts was determined by monitoring the transport of Rhodamine 6 G (R6G) across the cell membrane and IC50 values were obtained. Membrane permeabilizing properties of both extracts were also evaluated using the membrane potential sensitive dye 3'3 dipropylthiadicarbocyanine (diSC3-5). Haemolysis effect of both extracts on sheep erythrocytes was also investigated.

Results: Both extracts inhibited bacterial efflux pumps which resulted in the accumulation of R6G inside the cell. The IC50 values for C. citrinus and V. adoensis against S. aureus were 1.44 mg/ml and 1.61 mg/ml, respectively. Both leaf extracts however, showed similar IC50 values of 1.64 mg/ml against P. aeruginosa. Both plant extracts showed some significant effects on permeability of the bacterial membrane when a 24-28% increase of diSC3-5 dye release was observed for S. aureus and 45-53% of dye was released from P. aeruginosa cell membrane after a 60 minute incubation period. In addition, both extracts exhibited haemolytic effects on sheep erythrocytes at concentrations greater than 2.5 mg/ml.

Conclusions: These plant extracts may provide new lead compounds for developing potential efflux pump inhibitors (EPIs) or permeabilising agents that could aid the transport of antibacterial agents into bacterial cells.

Figure 1

R6G uptake and IC ₅₀ of efflux pump inhibition activity for A (reserpine), B (C. citrinus) and C ( V. adoensis) against P. aeruginosa.

Figure 2

R6G uptake and IC ₅₀ of efflux pump inhibition activity for A (reserpine), B ( C. citrinus) and C ( V. adoensis) against S. aureus.

Figure 3

Measurement of 3’3 dipropylthiadicarbocyanine (diSC3-5) dye release overtime from S. aureus (A) and P. aeruginosa (B) membranes in the presence of permeabilizing agent 1% DMSO, negative control ethanol and plant extracts C. citrinus and V. adoensis .

Figure 4

Extent of haemolysis of sheep erythrocytes with increasing concentration of plant extracts at the end of a 90 minute incubation period at 25°C (A) and 37°C (B).