Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms

Abstract

Staphylococcus aureus is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms. S. aureus bacteria in biofilms are protected from killing by antibiotics and the body's immune system. For years, one mechanism behind biofilm resistance to attack from the immune system's sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured S. aureus biofilm.

FIG. 1.

Digital time-lapse microscopy of human leukocytes interacting with a S. aureus biofilm (strain 1945). (A) Micrograph snapshot of leukocytes interacting with a 7-day-old S. aureus biofilm grown under flow conditions. Arrows identify biofilm-bound leukocytes. Note the large number of attached leukocytes in the pocket that are not present in panel B. (B) Micrograph snapshot of leukocytes interacting with the same biofilm as in panel A (∼1 h following leukocyte addition to the system). Note the reduction in leukocyte numbers attached to the surface of the biofilm later in the assay.

FIG. 2.

SCLM of human leukocytes interacting with an S. aureus biofilm. (A) Micrograph showing leukocytes (arrows) attached to and within an S. aureus biofilm. Note the presence of leukocytes and channels within the biofilm. (B and C). x (B) and Y (C) cross-sections, showing that the leukocytes have penetrated to the base of the biofilm. Bar, 20 μm.

FIG. 3.

High-magnification SCLM of a single representative leukocyte bound within an S. aureus biofilm. (A) Micrograph showing the “halo” of space surrounding the leukocyte. (B and C) Representative cross-sections (see Fig. 2 Legend). Bar, 10 μm.

FIG. 4.

Epifluorescence microscopy and SCLM of human leukocytes and GFP-expressing S. aureus. (A) Epifluorescence micrograph showing GFP-expressing S. aureus (planktonic) inside a human leukocyte. Note the numerous planktonic bacteria that have been engulfed (arrows). (B) SCLM of human leukocytes embedded within a GFP-expressing S. aureus (7-day-old) biofilm. (C) Higher magnification of a single human leukocyte that has penetrated the 7-day-old S. aureus biofilm. Note the absence of GFP-expressing S. aureus cells inside the representative leukocyte. Separate scale bars are listed for each micrograph.

FIG. 5.

(A) SLCM showing green-fluorescing leukocytes (L) attached to and within a statically grown, 2-day-old S. aureus biofilm (Sa). Note the presence of multilobed nucleated leukocytes within the biofilm. (B and C) x (B) and y (C) cross-sections showing that the leukocytes have not fully penetrated the biofilm but have simply sunk into the physical space that is present in the biofilm's three-dimensional structure. Bar, 30 μm.