Nitazoxanide inhibits biofilm formation by Staphylococcus epidermidis by blocking accumulation on surfaces

Abstract

Coagulase-negative species of Staphylococcus are often associated with opportunistic hospital-acquired infections that arise from the colonization of indwelling catheters. Here we show that the antiparasitic drug nitazoxanide (NTZ) and its active metabolite, tizoxanide (TIZ), are inhibitory to the growth of Staphylococcus epidermidis and other staphylococci, including methicillin-resistant Staphylococcus aureus strains, under aerobic and microaerobic conditions (MICs, 8 to 16 microg/ml). At sub-MIC levels, NTZ and TIZ also inhibited biofilm production under static conditions by strains of S. epidermidis and Staphylococcus haemolyticus with a 50% inhibitory concentration of approximately 2.5 microg/ml (8 microM). The 5-nitro group was required for biological activity, and a hydrophilic derivative of NTZ (AMIX) also inhibited biofilm formation. NTZ did not disperse the existing biofilm but did block further accumulation. Sub-MICs of NTZ had no effect on primary attachment to surfaces at either 4 or 37 degrees C. The inhibitory action of NTZ and TIZ, but not vancomycin, on biofilm production could be reversed by the addition of zinc salts (2.5 to 40 microM) but not other metals, suggesting that NTZ might target the zinc-dependent accumulation-associated protein (Aap) that mediates accumulation on surfaces. However, neither NTZ nor TIZ formed chelation complexes with zinc salts, based on spectrophotometric and nuclear magnetic resonance analyses, and addition of excess zinc to NTZ-grown bacteria (apo-Aap) did not restore the accumulation phenotype. Our studies suggest that sub-MIC levels of NTZ may affect the assembly or function of cell structures associated with the biofilm phenotype.

FIG. 1.

Chemical structures of NTZ and AMIX.

FIG. 2.

NTZ inhibition of S. epidermidis growth and biofilm production. (A) MIC testing. Biofilm-forming strain 9142 (▪) and non-biofilm-forming strains 5179 (⧫) and CAV1005 (•) were tested for their susceptibilities to NTZ by microdilution, and bacterial growth was measured by measurement of the turbidity, as described in the text. (B) Aerobic growth. Bacteria were grown in TSB medium with shaking at 37°C in the presence of no NTZ (⧫), 10 μg/ml NTZ (▪), or 25 μg/ml NTZ ▴). (C) Biofilm inhibition by NTZ. Staphylococcus strains were grown in the presence of different concentrations of NTZ in 96-well polystyrene plates and subjected to crystal violet staining after 24 h, as described in the text. The biofilm-positive strains (see the key) used were S. epidermidis 9142 and S. haemolyticus S33208, and the biofilm-negative strains were S. hominis F13532 and S. epidermidis CAV1005. The IC₅₀ for biofilm-producing strains was 2.5 μg/ml.

FIG. 3.

Screening for antibiofilm activity. (A) Clinical isolates confirmed to be S. epidermidis were tested for biofilm production and for concentration-dependent biofilm inhibition by NTZ at the indicated concentrations, with DMSO serving as an additional control. Strains ICS1 and ICS5 were biofilm positive. (B) Concentration-dependent inhibition of biofilm production of S. epidermidis strain 9142, icaA mutant strain 5179, and revertant strain 5179-R1, which produces a truncated Aap. The data presented represent the means and standard deviations of three replicates.

FIG. 4.

Effect of NTZ on attachment to catheter material. One-centimeter-square polyurethane pieces were incubated with the indicated concentrations of NTZ and S. epidermidis strain 9142, and the adherent bacteria were enumerated at 24 h. Means and standard deviations were determined from triplicates.

FIG. 5.

Biofilm dispersal by NTZ. S. epidermidis strain 9142 bacteria were grown under biofilm-producing conditions for 4 h. Following washing of the bacteria, fresh medium and the indicated concentrations of NTZ were added for an additional 16 h, and biofilm accumulation was determined with crystal violet. NTZ inhibited further biofilm formation in a concentration-dependent manner but did not disperse the existing biofilm.

FIG. 6.

Effect of zinc on bacterial growth and biofilm production. (A) Biofilm. Zinc sulfate (black bars), calcium chloride (hatched bars), and magnesium chloride (white bars) were added at the indicated concentrations to TSB containing a fixed concentration of NTZ (12.5 μg/ml; gray bars). Zinc chloride, but not the other metals, reversed the biofilm-inhibitory effect of NTZ, with 50% reversal occurring at ∼5 μM. (B) Growth. Effect of the following metals or conditions on bacterial growth at a fixed concentration of NTZ (12.5 μg/ml): ZnSO₄ (black bars), ZnCl₂ (dark gray bars), no metal (light gray bar), DMSO control (white bar), and no NTZ (dark gray bar).

FIG. 7.

Comparative antibiofilm activities of NTZ and AMIX. The inhibition of biofilm production by S. epidermidis strain 9142 by NTZ (•) and water-soluble AMIX (▪) was concentration dependent. All assays were performed in triplicate, with means and standard deviations being presented.