The ica locus of Staphylococcus epidermidis encodes production of the capsular polysaccharide/adhesin

Abstract

Clinical isolates of coagulase-negative staphylococci often elaborate a biofilm involved in adherence to medical devices and resistance to host defenses. The biofilm contains the capsular polysaccharide/adhesin (PS/A), which mediates cell adherence to biomaterials, and another antigen, termed polysaccharide intercellular adhesin (PIA), which is thought to mediate bacterial accumulation into cellular aggregates. PIA is a polymer of beta-1, 6-linked N-acetyl glucosamine residues with a molecular mass of <30, 000 kDa. We found that recombinant Staphylococcus carnosus and Staphylococcus aureus carrying a plasmid with genes of the ica locus, which was reported to encode the biosynthetic proteins for production of PIA, were also able to synthesize PS/A. PS/A and a chemically and immunologically identical polysaccharide isolated from S. carnosus carrying the ica genes on plasmid pCN27 were found to be high-molecular-mass (>250,000 kDa), acid-stable polymers of beta-1,6-linked glucosamine substituted on the amino group primarily with succinate, although some preparations also contained acetate. Moreover, all recombinant staphylococcal strains with the ica genes had the biologic properties previously attributed to PS/A. ica-positive strains readily formed an in vitro biofilm on plastic, adhered 3- to 10-fold more to catheters during a 30-min assay compared with control strains carrying only the cloning vector, adsorbed out antibodies to PS/A from immune serum, and elaborated a capsule visualized by immunoelectron microscopy with antisera to PS/A. These properties were also seen with PS/A-producing strains of Staphylococcus epidermidis, but not with transposon mutants lacking PS/A. An antiserum raised to PIA contained high-titer antibody to PS/A that was readily adsorbed out by PS/A-positive strains of S. epidermidis and recombinant strains of staphylococci carrying the ica genes. We conclude that the ica locus encodes production of PS/A and that the properties of S. epidermidis associated with initial bacterial adherence, biofilm formation, and intercellular adhesion can be correlated with elaboration of PS/A.

FIG. 1

ELISA inhibition with polyclonal antiserum to purified PS/A (A) and PIA (B) adsorbed with various staphylococcal strains. ELISA plates were coated with PS/A (2 μg/well). In all cases, significantly more antibody to PS/A was adsorbed by parental strains or strains carrying the ica locus than by the corresponding isogenic mutants or vector-control recombinant strains (P ≤ 0.005; unpaired t tests). Bars represent means, and error bars represent the standard errors of the means of five separate measurements.

FIG. 2

Plastic adherence (biofilm assay). Staphylococcal strains were assessed for their abilities to adhere to the plastic, as visualized by safranin staining. Bars represent means, and error bars represent the standard errors of the means for five separate measurements. Significant differences in adherence were noted between all parental strains and isogenic mutants, as well as between S. carnosus and S. aureus strains with the ica locus in plasmid pCN27 and control strains with plasmids lacking the ica locus (∗, P ≤ 0.0001 [t tests for comparison with isogenic strain; ∗∗, P < 0.001 [ANOVA and Fisher PLSD for pairwise comparisons with strain S. epidermidis M187]).

FIG. 3

Catheter adherence assay. After incubation for 30 min at 37°C, significant differences were noted between S. epidermidis parental strain M187 and isogenic mutant M187-sn3, S. carnosus(pCN27) and S. carnosus(pCA44), and S. aureus(pCN27) and S. aureus(pSK265) (∗, P ≤ 0.0001 [t tests]). Bars represent means, and error bars represent the standard errors of the means of five separate measurements.

FIG. 4

Immunoelectron microscopy of staphylococcal strains probed first with antiserum to PS/A and then with gold-labeled protein A (CoNS strains) or gold-labeled anti-rabbit IgG (S. aureus strains). (a) S. epidermidis M187; (b) S. epidermidis M187-sn3; (c) S. carnosus(pCN27); (d) S. carnosus(pCA44); (e) S. aureus(pCN27); (f) S. aureus(pSK265); (g) S. epidermidis 1457; (h) S. epidermidis 1457-M11; (i and j) S. epidermidis M187 and S. carnosus(pCN27), respectively, probed with normal rabbit serum. Bars in each panel, 200 nm.

FIG. 5

Immunoelectron microscopy of staphylococcal strains probed with antiserum to PIA and protein A-labeled gold. (a) S. epidermidis M187; (b) S. epidermidis M187-sn3; (c) S. carnosus(pCN27); (d) S. carnosus(pCA44); (e) S. epidermidis 1457; (f) S. epidermidis 1457-M11. Bars in each panel, 200 nm.