Epigallocatechin-gallate enhances the activity of tetracycline in staphylococci by inhibiting its efflux from bacterial cells

Abstract

Epigallocatechin-gallate (EGCg), the major catechin present in green tea extracts, has been shown to have several antibacterial activities, limiting bacterial growth and invasion and acting in synergy with beta-lactam antibiotics. In this article, we report that EGCg at doses half and below its calculated MIC of 100 microg/ml, is able to reverse tetracycline resistance in staphylococcal isolates expressing the specific efflux pump Tet(K) and appears to improve the MICs of tetracycline for susceptible staphylococcal isolates as well. The visible effect of EGCg is an increased accumulation of tetracycline inside bacterial cells. This effect is likely due to the inhibition of pump activity, and it is evident not only for Tet(K) pumps but also for efflux pumps of a different class [Tet(B)]. In summary, our data indicate that the observed dramatic enhancement by EGCg of tetracycline activity for resistant staphylococcal isolates is caused by impairment of tetracycline efflux pump activity and increased intracellular retention of the drug, suggesting a possible use of EGCg as an adjuvant in antibacterial therapy.

FIG. 1.

Time kill-curves for tet(K)-resistant (A) and tet(K)-susceptible (B) S. epidermidis. Bacteria were grown in the presence of one-half and one-fourth the MICs of tetracycline (32 and 16 μg/ml for resistant strains and 0.5 and 0.25 μg/ml for susceptible strains, respectively), with and without EGCg at 50 μg/ml (corresponding to one-half the MIC). Symbols: ○, control; •, tetracycline (one-half the MIC); ▪, tetracycline (one-fourth the MIC); □, EGCg at 50 μg/ml (one-half the MIC); ▴, tetracycline (one-half the MIC) plus EGCg at 50 μg/ml; ⋄, tetracycline (one-fourth the MIC) plus EGCg at 50 μg/ml. Error bars indicate standard deviations.

FIG. 2.

Dynamics of tetracycline uptake and release. Intact tetracycline-resistant or tetracycline-susceptible S. epidermidis bacterial cells (A) or resistant S. epidermidis cells stripped of the cell wall (B) were incubated with tetracycline (100 μg/ml), with or without EGCg preincubation (50 μg/ml), in Mg²⁺ buffer. Under these conditions, tetracycline that enters the cells becomes fluorescent, and once extruded from the cell, it can be detected and quantitated with a spectrofluorophotometer. The curves shown thus represent the relative amounts of tetracycline pumped out of the cells under the various incubation conditions.

FIG. 3.

Tetracycline accumulation and release by Tet(K)-resistant and Tet(K)-susceptible S. epidermidis strains. Bacterial suspensions were incubated in triplicate for 15 min with tetracycline (100 μg/ml), with or without EGCg preincubation (50 μg/ml). Under these conditions, tetracycline is dynamically accumulated within the cells, depending on the activity of their efflux pumps. After a rapid rinsing, the cells are resuspended in Mg²⁺ buffer, in which released fluorescence (a direct measurement of accumulated tetracycline) is immediately detected with a spectrofluorophotometer. From top to bottom, the bars represent tetracycline-resistant cells, tetracycline-resistant cells in the presence of EGCg, tetracycline-susceptible cells, and tetracycline-susceptible cells in the presence of EGCg. Error bars indicate standard deviations.