Comparative antibody-mediated phagocytosis of Staphylococcus epidermidis cells grown in a biofilm or in the planktonic state

Abstract

Staphylococcus epidermidis is an important cause of nosocomial infections. Virulence is attributable to elaboration of biofilms on medical surfaces that protect the organisms from immune system clearance. Even though leukocytes can penetrate biofilms, they fail to phagocytose and kill bacteria. The properties that make biofilm bacteria resistant to the immune system are not well characterized. In order to better understand the mechanisms of resistance of bacteria in biofilms to the immune system, we evaluated antibody penetration throughout the biofilm and antibody-mediated phagocytic killing of planktonic versus biofilm cells of S. epidermidis by using a rabbit antibody to poly-N-acetylglucosamine (PNAG). These antibodies are opsonic and protect against infection with planktonic cells of PNAG-positive Staphylococcus aureus and S. epidermidis. Antibody to PNAG readily penetrated the biofilm and bound to the same areas in the biofilm as did wheat germ agglutinin, a lectin known to bind to components of staphylococcal biofilms. However, biofilm cells were more resistant to opsonic killing than their planktonic counterparts in spite of producing more PNAG per cell than planktonic cells. Biofilm extracts inhibited opsonic killing mediated by antibody to PNAG, suggesting that the PNAG antigen within the biofilm matrix prevents antibody binding close to the bacterial cell surface, which is needed for efficient opsonic killing. Increased resistance of biofilm cells to opsonic killing mediated by an otherwise protective antibody was due not to a biofilm-specific phenotype but rather to high levels of antigen within the biofilm that prevented bacterial opsonization by the antibody.

FIG. 1.

Analysis of the ability of rabbit IgG antibodies to PNAG to penetrate into a biofilm formed by S. epidermidis strain 9142; the x axis (upper panel) and y axis (lower panel) cross sections of the biofilm are shown. Rabbit antibody to PNAG was visualized using goat anti-rabbit IgG conjugated to Alexa 488.

FIG. 2.

Analysis of binding of either rabbit IgG to PNAG or WGA to components of the biofilm formed by S. epidermidis strain 9142. The following Z sections are shown: panel A, binding of rabbit IgG to PNAG visualized by a goat antibody to rabbit IgG conjugated to Alexa 488; panel B, WGA directly conjugated to Texas Red; panel C, overall biofilm structure as visualized by the refraction of far red light; panel D, colocalization of binding of antibody to PNAG and WGA. In panel D, the antibody signal is shown in green and the lectin signal is shown in red, with the overlap causing the yellow color.

FIG. 3.

Opsonophagocytic killing of different S. epidermidis stains grown either in the planktonic (P) or biofilm (B) mode. Killing was determined using a 1:10 dilution of rabbit antibody raised to deacetylated PNAG conjugated to diphtheria toxoid, and the percent killing was calculated using the mean CFU in duplicate determinations from three different control tubes lacking one of the assay components as the denominator. Killing in the presence of normal rabbit serum was always <5%. There were no significant differences in killing achieved when comparing all four strains grown as planktonic cells and no significant differences in killing when comparing all four strains grown as biofilm cells (P > 0.1; ANOVA). There were significant differences in killing for each strain when comparing planktonic versus biofilm cells at P < 0.05 (unpaired samples t test).

FIG. 4.

Expression of PNAG in culture supernatants and cell surface extracts of S. epidermidis strain 9142. Dilutions of each sample are indicated on the top of the figure. Row 1, supernatant from planktonic cells; row 2, EDTA extract of planktonic cells; row 3, supernatant of biofilm cells; row 4, EDTA cell extract of biofilm cells.

FIG. 5.

Inhibition of opsonic killing of planktonic cells of S. epidermidis strain 9142. A rabbit antiserum to deacetylated PNAG (diluted 1:10) was adsorbed with either TSB, strain 9142 biofilm matrix (undiluted or diluted 1:10), strain 9142-M10 cell surface extract, or purified PNAG before use in the opsonophagocytic assay. The reduction in percent killing by both the biofilm matrix (undiluted and at 1:10 dilutions) and 50 mg/liter PNAG was significant at P < 0.05 (t test) when compared with the TSB-inhibited sample.