eap Gene as novel target for specific identification of Staphylococcus aureus

Abstract

The cell surface-associated extracellular adherence protein (Eap) mediates adherence of Staphylococcus aureus to host extracellular matrix components and inhibits inflammation, wound healing, and angiogenesis. A well-characterized collection of S. aureus and non-S. aureus staphylococcal isolates (n = 813) was tested for the presence of the Eap-encoding gene (eap) by PCR to investigate the use of the eap gene as a specific diagnostic tool for identification of S. aureus. Whereas all 597 S. aureus isolates were eap positive, this gene was not detectable in 216 non-S. aureus staphylococcal isolates comprising 47 different species and subspecies of coagulase-negative staphylococci and non-S. aureus coagulase-positive or coagulase-variable staphylococci. Furthermore, non-S. aureus isolates did not express Eap homologs, as verified on the transcriptional and protein levels. Based on these data, the sensitivity and specificity of the newly developed PCR targeting the eap gene were both 100%. Thus, the unique occurrence of Eap in S. aureus offers a promising tool particularly suitable for molecular diagnostics of this pathogen.

FIG. 1.

Schematic representation (not to scale) of Eap and the encoding gene, showing the amino acid (aa) composition consisting of a 30-aa signal peptide (S) and 110-aa residue domains (R1, R2, … RX) found in multiple copies. The oligonucleotide primer binding sites of EAP-CON1 (A1) and EAP-CON2 (A2) as well as of EAP-P3wt (B1) and EAP-P2wt (B2) are indicated in base pairs (bp); sizes correspond to EAP of the Newman D2C strain.

FIG. 2.

Agarose gel (1%) electrophoresis patterns showing PCR products amplified with eap primers by use of genomic DNA of S. aureus ATCC 29213 (lanes 1 and 4), S. aureus Newman (lanes 2 and 5), and S. aureus Wood 46 (lanes 3 and 6) as well as genomic DNA of S. epidermidis DSM 20044 (lane 7), S. haemolyticus DSM 20263 (lane 8), S. hominis subsp. hominis DSM 20238 (lane 9), S. intermedius DSM 20373 (lane 10), and S. lugdunensis DSM 4804 (lane 11). Lane M, DNA molecular mass marker (1-kb/100-bp DNA ladder); lanes 1 to 3, eap gene amplified with primers EAP-P2wt and EAP-P3wt; lanes 4 to 11, eap gene amplified with newly designed primers EAP-CON1 and EAP-CON2 (see Table 2).

FIG. 3.

Southern blot analysis of genomic DNA (A) and Northern blot of total RNA (B). Lanes 1 to 4, S. epidermidis; lane 5, S. carnosus subsp. carnosus; lane 6, S. haemolyticus; lane 7, S. hominis subsp. hominis; lanes 8 to 9, S. saprophyticus subsp. saprophyticus; lanes 10 to 12, S. aureus (lane 10, strain Newman; lane 11, strain CI7; lane 12, strain Wood 46). Blots were prepared as described in Materials and Methods.

FIG. 4.

SDS-PAGE (A) and Western immunoblot (B) analysis of SDS surface protein extracts. Lane M, marker; lanes 1 to 3, S. aureus strains CI7 (lane 1), Wood 46 (lane 2), and Newman (lane 3); lanes 4 to 8, strains of non-S. aureus staphylococcal species S. epidermidis (lanes 4 and 5), S. carnosus subsp. carnosus (lane 6), S. haemolyticus (lane 7), and S. hominis subsp. hominis (lane 8). Blots were prepared as described in Materials and Methods.