Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162 - PubMed (original) (raw)

Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162

Esther Heikens et al. J Bacteriol. 2007 Nov.

Abstract

Enterococci have emerged as important nosocomial pathogens with resistance to multiple antibiotics. Adhesion to abiotic materials and biofilm formation on medical devices are considered important virulence properties. A single clonal lineage of Enterococcus faecium, complex 17 (CC17), appears to be a successful nosocomial pathogen, and most CC17 isolates harbor the enterococcal surface protein gene, esp. In this study, we constructed an esp insertion-deletion mutant in a clinical E. faecium CC17 isolate. In addition, initial adherence and biofilm assays were performed. Compared to the wild-type strain, the esp insertion-deletion mutant no longer produced Esp on the cell surface and had significantly lower initial adherence to polystyrene and significantly less biofilm formation, resulting in levels of biofilm comparable to those of an esp-negative isolate. Capacities for initial adherence and biofilm formation were restored in the insertion-deletion mutant by in trans complementation with esp. These results identify Esp as the first documented determinant in E. faecium CC17 with an important role in biofilm formation, which is an essential factor in infection pathogenesis.

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Figures

FIG. 1.

Confirmation of correct insertion-deletion mutation in the esp gene by Southern blot analysis. (A) Schematic representation of the 5′ end encoding the N-terminal domain of the wild-type esp gene (1), the esp single-crossover insertion (2), and the esp double-crossover insertion-deletion (3). The box with squares represents the cat gene coding for chloramphenicol resistance, the striped box represents the aph2‴-Id gene coding for gentamicin resistance, the hatched box represents the EspUp fragment used for recombination, the stippled box represents the EspDn fragment used for recombination, and the black line represents the DNA probe. a, TaqI restriction site; b, fragments obtained after digestion with TaqI; c, nucleotide reference positions relative to the E. faecium PAI sequence (GenBank accession no. AY322150). (B) Hybridization results of Southern blot analysis of TaqI-digested genomic DNA of the esp wild-type strain (lane 1), esp single-crossover mutant strain (lane 2), and esp double-crossover mutant strain (lane 3).

FIG. 2.

Cell surface expression of Esp by flow cytometry. Shown is analysis of cell surface expression of Esp by flow cytometry using rabbit anti-Esp immune serum for the esp wild-type strain (E1162), esp mutant strain (E1162Δ_esp_), _esp_-negative strain (E135), and _esp_-complemented strain. Mean values and standard deviations are shown. *, P < 0.001; **, P < 0.0005.

FIG. 3.

Cell surface expression of Esp by whole-cell ELISA. Shown is analysis of cell surface expression of Esp by whole-cell ELISA using rabbit anti-Esp IgGs in different dilutions for the esp wild-type strain (E1162) (squares), esp mutant strain (E1162Δ_esp_) (triangles), _esp_- negative strain (E135) (diamonds), and esp_-complemented strain (E1162Δ_esp:pEF3) (circles). Mean values and standard deviations are shown.

FIG. 4.

Electron microscopy. Shown are electron micrographs at a magnification of ×60,000. The esp wild-type strain (E1162) (A) and the esp mutant strain (E1162Δ_esp_) (B) were incubated with rabbit anti-Esp immune serum, followed by protein-A-Gold. Bars, 200 nm.

FIG. 5.

Initial adherence and biofilm formation. Shown are the abilities to adhere to polystyrene (A) and to form biofilm (B) of the esp wild-type strain (E1162), esp mutant strain (E1162Δ_esp_), _esp_-negative strain (E135), and _esp_-complemented strain (E1162Δ_esp:_pEF3). The horizontal lines represent background OD levels when wells possessing no bacteria were stained with crystal violet. Mean values and standard deviations are shown. **, P < 0.0005, and ***, P < 0.0001 (A); *, P < 0.001, and **, P < 0.0005 (B).

FIG. 6.

CLSM images of the esp wild-type strain (E1162) (A) and the esp mutant strain (E1162Δ_esp_) (B) grown on nitrocellulose for 24 h. The images represent the layer in a Z-stack that has the maximum bacterial coverage. The maximum thickness of biofilms was measured at five randomly chosen positions, resulting in a mean maximum thickness of 11.01 (± 0.91) μm for E1162 and 6.43 (± 0.81) μm for E1162Δ_esp_. This difference was significant (P < 0.0001).

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Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162 - PubMed (original) (raw)