Iron blocks the accumulation and activity of tetracyclines in bacteria

Abstract

The apparent sensitivities of several bacterial pathogens to tetracyclines varied by up to 128-fold with the medium content of Fe, but not of other metals. The effect of Fe was independent of superoxide dismutase activity and of intracellular Fe, but accumulation of tetracyclines was blocked in high-Fe medium. Thus, synergistic suppression of bacterial growth in the presence of a low Fe concentration and tetracyclines arises because of elevated antibiotic accumulation.

FIG. 1.

Activities of TCs in the presence of iron. E. coli, S. aureus, or P. aeruginosa suspensions were pin inoculated into LB agar supplemented with a range of iron (FeSO₄) and antibiotic concentrations (the antibiotics were supplied in twofold dilution series). MICs were determined at each FeSO₄ concentration as the lowest antibiotic concentrations that resulted in full inhibition of visible growth in replicate incubations. (A) MICs of OTC (○),TC (•), and MC (□) for E. coli GC4468. (B) MICs of OTC for E. coli (○), S. aureus 8325-4 (•), and P. aeruginosa PAO1 (□). These results were reproduced in several independent experiments.

FIG. 2.

Qualitative analysis of OTC accumulation in the absence and presence of iron. Exponential-phase E. coli cultures were incubated for 15 min at 37°C and 200 rpm in LB broth in the absence (B) or presence (C to E) of 64 μg of OTC ml⁻¹ and with 50 μM Cu(NO₃)₂ (D) or 3 mM FeSO₄ (E) added. Cellular fluorescence from OTC was visualized under oil immersion with UV excitation with a Zeiss HBO-50/Ac upright fluorescence microscope. A 10× eyepiece and a 100× objective were used for examination. Images were captured with Axiovision 3.0 (Zeiss) software. The phase-contrast light image (A) shows the typical bacterial density in each field of view. Typical results from one of several independent experiments are shown. Similar results were obtained for S. aureus and P. aeruginosa (not shown).

FIG. 3.

Quantitative determination of OTC accumulation in the absence and presence of iron. Exponential-phase E. coli cultures were incubated in LB broth with 64 μg of OTC ml⁻¹ in the absence (○) and presence (•) of 3 mM FeSO₄. Samples were removed at intervals, and cells were separated by filtration. Supernatants were retained at 4°C until analysis. OTC in the supernatants was determined spectrophotometrically (3, 12). Cellular OTC accumulation was calculated by subtraction from OTC determinations for control incubations lacking cells. The points are means of three replicate determinations from each of two independent experiments ± the standard errors of the means where these exceed the dimensions of the symbols.