Abolition of biofilm formation in urinary tract Escherichia coli and Klebsiella isolates by metal interference through competition for fur

Abstract

Bacterial biofilms are associated with a large number of persistent and chronic infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics and immune defenses, which makes it hard if not impossible to eradicate biofilm-associated infections. In the urinary tract, free iron is strictly limited but is critical for bacterial growth. Biofilm-associated Escherichia coli cells are particularly desperate for iron. An attractive way of inhibiting biofilm formation is to fool the bacterial regulatory system for iron uptake. Here, we demonstrate that biofilm formation can be impaired by the addition of divalent metal ions, such as Zn(II) and Co(II), which inhibit iron uptake by virtue of their higher-than-iron affinity for the master controller protein of iron uptake, Fur. Reduced biofilm formation of urinary tract-infectious E. coli strains in the presence of Zn(II) was observed in microtiter plates and flow chambers as well as on urinary catheters. These results further support that iron uptake is indeed crucial for biofilm formation, and thereby, targeting these uptake systems might be an effective way to eradicate biofilms caused by infectious strains.

FIG. 1.

Biofilm formation in microtiter plates of E. coli and Klebsiella sp. urinary tract isolates in minimal laboratory medium in the presence of Zn(II) (ZnSO₄) and Co(II) [Co(NO₃)₂]. Values are shown relative to strains forming biofilms in the same medium without the addition of metal ions. Biofilm formation was determined by crystal violet staining. Values are means of at least three independent experiments, and error bars indicate standard deviations (σ_{n − 1}). All strains showed significantly reduced biofilm formation in the presence of Zn(II) or Co(II) (P < 0.001 by paired t test). Zn(II), Co(II), and the two metals in combination were tested for nine, six, and three of the strains, respectively.

FIG. 2.

Addition of Zn(II) inhibits biofilm formation of UTI strain E. coli 83972 in flow chambers (A) and strain VR50 on urinary catheters (B). (A) Biofilm formation of strain 83972yfp in minimal medium with and without 50 μM ZnSO₄ was examined in the flow cell chamber system and monitored by using scanning confocal laser microscopy at 16, 24, and 40 h postinoculation. Scale bars represent 50 μm. (B) Biofilm formation of strain VR50 in minimal medium with and without 500 μM ZnSO₄ was examined on urinary catheters (100% silicone) by viable counts. Catheters were incubated for 24 h, washed, and sonicated, and serial dilutions were plated for determinations of CFU. Results displayed are means of five replicates, and error bars indicate standard deviations (σ_{n− 1}). VR50 formed significantly less biofilm on catheters in medium with Zn(II) than in medium with no additional zinc ions (P < 0.01 by t test).

FIG. 3.

Biofilm formation in microtiter plates of the two E. coli UTI strains 83972 and VR50 in human urine with and without the addition of iron (10 μM FeCl₃), zinc (ZnSO₄), and cobalt [Co(NO₃)₂]. Values in the presence of metal ions are shown relative to those of the control (i.e., strain grown in urine without the addition of metal ions). Biofilm formation was determined by crystal violet staining. Values are means of at least three independent experiments, and error bars indicate standard deviations (σ_{n− 1}). All values were significantly different from those of the control (P < 0.001 by paired t test).

FIG. 4.

Biofilm formation of E. coli UTI strain 83972 overexpressing Fur in iron-limiting minimal medium with and without the addition of zinc (ZnSO₄) and iron (FeCl₃). Biofilm formation is shown relative to that of the control (i.e., native strain grown without the addition of metal ions) and was determined by crystal violet staining. Values are means of seven independent experiments, and error bars indicate standard deviations (σ_{n− 1}). Asterisks indicate significant differences in biofilm formation. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (paired t test).