Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci

Abstract

Extracellular DNA is an adhesive component of staphylococcal biofilms. The aim of this study was to evaluate the antibiofilm activity of recombinant human DNase I (rhDNase) against Staphylococcus aureus and Staphylococcus epidermidis. Using a 96-well microtiter plate crystal-violet binding assay, we found that biofilm formation by S. aureus was efficiently inhibited by rhDNase at 1-4 μg l⁻¹, and preformed S. aureus biofilms were efficiently detached in 2 min by rhDNase at 1 mg l⁻¹. Pretreatment of S. aureus biofilms for 10 min with 10 mg l⁻¹ rhDNase increased their sensitivity to biocide killing by 4-5 log units. rhDNase at 10 mg l⁻¹ significantly inhibited biofilm formation by S. epidermidis in medium supplemented with sub-MICs of antibiotics. We also found that rhDNase significantly increased the survival of S. aureus-infected Caenorhabditis elegans nematodes treated with tobramycin compared with nematodes treated with tobramycin alone. We concluded that rhDNase exhibits potent antibiofilm and antimicrobial-sensitizing activities against S. aureus and S. epidermidis at clinically achievable concentrations. rhDNase, either alone or in combination with antimicrobial agents, may have applications in treating or preventing staphylococcal biofilm-related infections.

Figure 1

Inhibition (a) and detachment (b) of S. aureus biofilms by rhDNase in a 96-well microtiter plate crystal violet binding assay. (a) The indicated strains were cultured in TSB supplemented with increasing concentrations of rhDNase. Absorbance at 595 nm is proportional to biofilm biomass. (b) Biofilms were cultured for 18 h in unsupplemented TSB, rinsed with water, and then treated with rhDNase (1 mg l⁻¹) for increasing amounts of time. For panels (a) and (b), values represent the means for duplicate wells. Error bars were omitted for clarity.

Figure 2

Pre-treatment of S. aureus biofilms with rhDNase renders them sensitive to killing by topical biocides. S. aureus SH1000 biofilms grown in 96-well microtiter plates were treated with the indicated agent for 5 min. In some wells, biofilms were pre-treated with 10 mg l⁻¹ rhDNase for 10 min prior to the addition of the agent. Values show mean numbers of surviving CFU/well, and error bars indicate range.

Figure 3

Killing of the nematode C. elegans by S. aureus in the presence of tobramycin and rhDNase. The graph shows Kaplan-Meier survival curves of uninfected nematodes, nematodes infected with S. aureus KBI-11, and nematodes infected with S. aureus KBI-11 and cultured in the presence of 2.5 mg l⁻¹ rhDNase, 1.25 mg l⁻¹ tobramycin, or 2.5 mg l⁻¹ rhDNase plus 1.25 mg l⁻¹ tobramycin.

Figure 4

Inhibition of S. epidermidis biofilm formation by rhDNase and dispersin B in 96-well microtiter plates. S. epidermidis strain RP62A was cultured in the presence of 10 mg l⁻¹ rhDNase, 10 mg l⁻¹ dispersin B (DspB), or a combination of both enzymes at 10 mg l⁻¹ each. Some wells were further supplemented with 1.5 mg l⁻¹ vancomycin (2/3 × MIC), 0.02 mg l⁻¹ tigecycline (1/4 × MIC). After 16 h, biofilms were rinsed with water and quantitated by crystal violet staining as described in the legend to Figure 1. Values show mean absorbance values for duplicate wells, and error bars indicate range.