Current concepts in biofilm formation of Staphylococcus epidermidis

Abstract

Staphylococcus epidermidis is a highly significant nosocomial pathogen mediating infections primarily associated with indwelling biomaterials (e.g., catheters and prostheses). In contrast to Staphylococcus aureus, virulence properties associated with S. epidermidis are few and biofilm formation is the defining virulence factor associated with disease, as demonstrated by animal models of biomaterial-related infections. However, other virulence factors, such as phenol-soluble modulins and poly-gamma-DL-glutamic acid, have been recently recognized that thwart innate immune system mechanisms. Formation of S. epidermidis biofilm is typically considered a four-step process consisting of adherence, accumulation, maturation and dispersal. This article will discuss recent advances in the study of these four steps, including accumulation, which can be either polysaccharide or protein mediated. It is hypothesized that studies focused on understanding the biological function of each step in staphylococcal biofilm formation will yield new treatment modalities to treat these recalcitrant infections.

Figure 1. Biofilm formation by Staphylococcus epidermidis 1457 and an isogenic icaADBC mutant as compared in a flow cell

(A–C) S. epidermidis 1457 icaADBC::dhfr [69] grown in a flow cell for 24 (A), 48 (B) and 72 h (C). (D–F) Staphylococcus epidermidis 1457 [51] grown in a flow cell for 24 (D), 48 (E) and 72 h (F). Note the significant tower formation and 3D structure associated with 1457 as compared with 1457 icaADBC at 48 and 72 h (noted by arrow in (E)). S. epidermidis 1457 sarA::tetM [69] (G), S. epidermidis 1457 icaADBC::dhfr (H) and S. epidermidis 1457 (I) grown in a flow cell with high shear stress. Note the lack of biofilm formation in (G & H) containing S. epidermidis 1457 mutants unable to synthesize polysaccharide intercellular adhesin (polysaccharide intercellular adhesin [PIA]; icaADBC and sarA mutations). Tryptic soy broth at a flow rate of 0.5 ml/min was used in both flow cells shown in (A–F) and (G–I). However, the shear stress was greater in the flow cell shown in panels (G–I) due to the smaller surface area of the material supporting bacterial growth. (J & K) Note the contribution of PIA to biofilm synthesis in 1457 (J) in contrast to 1457 icaADBC::dhfr (K). S. epidermidis 1457 PIA-dependent biofilms can be picked up with a pipette, whereas PIA-independent biofilms in the 1457 background can easily be resuspended with a pipette. (L–N) Note that biofilms from 72 h flow cells (as shown in (C & F)) from 1457 icaADBC::dhfr (M) and 1457 sarA::tetM can easily be resuspended in broth, whereas the biofilm from 1457 (L) is not resuspended upon vortexing.

Figure 2. Confocal microscopy of polysaccharide intercellular adhesin-dependent and -independent biofilms

Staphylococcus epidermidis 1457 icaADBC::dhfr (A) and 1457 (B) were grown in Lab-Tek^™ borosilicate coverglass systems for 24 h in tryptic soy broth and stained with wheat germ agglutinin (WGA), Syto9 and Toto-3. WGA (purple stain) was used to identify polysaccharide intercellular adhesin, Syto-9 (green stain) was applied to identify viable cells in the biofilm, and Toto-3 (red stain) was used to stain both dead cells and eDNA. Note that, in contrast to towers found in 1457, the towers in 1457 icaADBC::dhfr are comprised of dead cells and/or eDNA. 1457 towers are much more common, larger and contain live cells. In addition, note the WGA staining of polysaccharide intercellular adhesin in 1457 (B).

Figure 3. Temporal and spatial heterogeneity in a staphylococcal biofilm

(A) Young biofilm replete with oxygen and nutrient substrate. By contrast, mature biofilm has cells that have access to both oxygen and substrate (B), substrate but no oxygen (C) and no oxygen or substrate (D), generating metabolic heterogeneity. In media containing a readily catabolizeable substrate such as glucose and in addition a separate carbon source such as amino acids or peptides, the upper regions of the biofilm (B) would have access to the glucose, whereas more microaerobic regions (C) would have access to a secondary carbon source such as amino acids/peptides. Adapted from [94].

Figure 4. Link between biofilm maturation and phenotypic variation

Flow cell biofilms of Staphylococcus epidermidis 1457 were grown in tryptic soy broth for 24 (A), 48 (B) and 72 h (C), and plated onto Congo red agar. Phenotypic variants (as noted by arrows) are readily observed coincident with tower formation and biofilm maturation. Note no phenotypic variants or towers were observed after 24 h of growth (authors’ observation and [114]).