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, 48 (7), 2633-6

Enzymatic Detachment of Staphylococcus Epidermidis Biofilms

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Enzymatic Detachment of Staphylococcus Epidermidis Biofilms

Jeffrey B Kaplan et al. Antimicrob Agents Chemother.

Abstract

The gram-positive bacterium Staphylococcus epidermidis is the most common cause of infections associated with catheters and other indwelling medical devices. S. epidermidis produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces. We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans rapidly and efficiently removed S. epidermidis biofilms from plastic surfaces. The enzyme worked by releasing extracellular slime from S. epidermidis cells. Precoating surfaces with the enzyme prevented S. epidermidis biofilm formation. Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents.

Figures

FIG. 1.
FIG. 1.
S. epidermidis biofilm formation in the wells of 96-well polystyrene microtiter plates. (A) Serial decimal dilutions (bottom axis) of four S. epidermidis strains (left axis) were grown in duplicate horizontal rows of wells and then rinsed with water and stained with crystal violet. Thirty minutes prior to rinsing, dispersin B was added to alternate horizontal rows of wells (right axis). (B) OD of the wells in panel A. Filled circles correspond to values for untreated rows. Open circles correspond to values for dispersin B-treated rows. (C) S. epidermidis biofilms were mock treated (Control) or treated with dispersin B or heat-inactivated dispersin B and then stained with crystal violet. (D) S. epidermidis biofilms were treated with increasing amounts of A. actinomycetemcomitans dispersin B, S. marcescens chitinase, or Jack bean N-acetylglucosaminidase (NAGase) and then rinsed with water and stained with crystal violet.
FIG. 2.
FIG. 2.
Time course and dose-response curves for dispersin B-mediated detachment of S. epidermidis biofilms from the wells of a 96-well microtiter plate. (A) Biofilms were treated with increasing amounts of dispersin B (vertical axis) for increasing amounts of time (horizontal axis). The wells were then rinsed with water and stained with crystal violet. (B) OD of the rows of wells in panel A corresponding to 0.04 μg ml−1 (filled squares), 0.2 μg ml−1 (open squares), 1.0 μg ml−1 (filled circles), and 4.8 μg ml−1 (open circles).
FIG. 3.
FIG. 3.
Growth of S. epidermidis biofilms on the surfaces of polystyrene rods and polyurethane and Teflon intravenous catheters. (A) Polystyrene rods (1.5-mm diameter) stained with crystal violet. Rods were incubated in broth containing S. epidermidis strain NJ9709 (rods 1 to 3) or in uninoculated broth (rod 4). Prior to staining, rod 1 was mock treated, rod 2 was treated with dispersin B, and rod 3 was sonicated. (B) The bacteria remaining attached to the polystyrene rods after mock treatment and treatment with dispersin B were removed by sonication and enumerated by plating of serial dilutions on agar. Values indicate mean numbers of bacteria per rod (± standard error) for triplicate rods. (C) Polyurethane catheters were incubated in broth containing S. epidermidis strain NJ9709 and then mock treated (left catheter) or treated with dispersin B (right catheter). Catheters were then rinsed and stained with methylene blue. (D) Teflon catheters (1.2-mm diameter) were treated as described for panel C, except that bacteria were stained with crystal violet. (E) Polyurethane catheters were precoated with PBS (left catheter) or PBS plus dispersin B (right catheter), rinsed with water, and then incubated in broth containing S. epidermidis strain NJ9709 for an additional 6 h. Catheters were then rinsed and stained with methylene blue. (F) Teflon catheters were treated as described for panel E, except that bacteria were stained with crystal violet.
FIG. 4.
FIG. 4.
Dispersin B releases extracellular slime from S. epidermidis biofilm cells. Minus sign, mock-treated cells; plus sign, dispersin B-treated cells. (A) Biofilm cells that were rinsed and scraped from the surface of a culture vessel formed an aggregate that settled to the bottom of the tube (left). Treatment of the cell aggregate with dispersin B for 5 min resulted in complete dispersion of the aggregate (right). (B) Quantitation of glycosaminoglycans in cell culture supernatants. The OD is proportional to the amount of glycosaminoglycans in the supernatants. Values show means (± standard error) for triplicate samples. (C) Quantitation of total hexosamine in cell culture supernatants. Values show means (± standard error) for triplicate samples. (D) Polysaccharides purified from cell culture supernatants were analyzed by polyacrylamide gel electrophoresis and stained with silver. The sizes (in kilodaltons) of molecular mass standards electrophoresed in an adjacent lane are indicated on the right.

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