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Journal of Clinical Microbiology
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Bacteriology

eap Gene as Novel Target for Specific Identification of Staphylococcus aureus

Muzaffar Hussain, Christof von Eiff, Bhanu Sinha, Insa Joost, Mathias Herrmann, Georg Peters, Karsten Becker
Muzaffar Hussain
1Institute of Medical Microbiology, University of Münster, 48149 Münster
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Christof von Eiff
1Institute of Medical Microbiology, University of Münster, 48149 Münster
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Bhanu Sinha
2Institute of Hygiene and Microbiology, University of Würzburg, 97080 Würzburg
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Insa Joost
3Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, 66421 Homburg/Saar, Germany
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Mathias Herrmann
3Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, 66421 Homburg/Saar, Germany
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Georg Peters
1Institute of Medical Microbiology, University of Münster, 48149 Münster
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Karsten Becker
1Institute of Medical Microbiology, University of Münster, 48149 Münster
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  • For correspondence: kbecker@uni-muenster.de
DOI: 10.1128/JCM.01425-07
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  • FIG. 1.
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    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.
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    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.
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    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.
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    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.

Tables

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  • TABLE 1.

    Non-S. aureus staphylococcal (sub-)species studied

    Species and subspeciesNo. of clinical isolatesaType and/or reference strain(s)bTotal no. of isolatesc
    BloodOther
    S. arlettae 00DSM 20672T1
    S. auricularis 00DSM 20609T1
    S. capitis subsp. capitis41DSM 20326T6
    S. capitis subsp. urealyticus00DSM 6717T1
    S. caprae 05DSM 20608T6
    S. carnosus subsp. carnosus00DSM 20501T1
    S. carnosus subsp. utilis00DSM 11676T1
    S. chromogenes 00DSM 20454T1
    S. cohnii subsp. cohnii42DSM 20260T7
    S. cohnii subsp. urealyticus00DSM 6718T1
    S. condimenti 00DSM 11674T1
    S. delphini 00DSM 20771T1
    S. epidermidis 455DSM 20044T51
    S. equorum subsp. equorum00DSM 20674T, DSM 206752
    S. equorum subsp. linens00DSM 15097T1
    S. felis 00DSM 7377T1
    S. fleurettii 00DSM 13212T1
    S. gallinarum 00DSM 20610T1
    S. haemolyticus 141DSM 20263T16
    S. hominis subsp. hominis90DSM 20328T10
    S. hominis subsp. novobiosepticus00ATCC 700236T1
    S. hyicus 01DSM 20459T2
    S. intermedius 110DSM 20373T12
    S. kloosii 00DSM 20676T1
    S. lentus 00DSM 20352T1
    S. lugdunensis 119DSM 4804T21
    S. lutrae 00DSM 10244T1
    S. muscae 00DSM 7068T1
    S. nepalensis 00DSM 15150T, DSM 151512
    S. pasteuri 00DSM 10656T1
    S. pettenkoferi 41CCUG 51270T6
    S. piscifermentans 00DSM 7373T1
    S. pseudintermedius 00LMG 22219T, LMG 22220, LMG 22221, LMG 222224
    S. saccharolyticus 00DSM 20359T1
    S. saprophyticus subsp. bovis00CCM 4410T1
    S. saprophyticus subsp. saprophyticus212DSM 20229T15
    S. schleiferi subsp. coagulans00DSM 6628T1
    S. schleiferi subsp. schleiferi05DSM 4807T6
    S. sciuri subsp. carnaticus00ATCC 700058T1
    S. sciuri subsp. rodentium00ATCC 700061T1
    S. sciuri subsp. sciuri21DSM 20345T4
    S. simulans 14DSM 20322T6
    S. succinus subsp. casei00DSM 15096T1
    S. succinus subsp. succinus00DSM 14617T1
    S. vitulinus 00ATCC 51145T1
    S. warneri 50DSM 20316T6
    S. xylosus 14DSM 20266T6
    Total937152216
    • ↵ a Isolates from blood and from other specimens (skin, wound, respiratory tract, urogenital tract, and venous catheters).

    • ↵ b Type or reference strains of the respective (sub-)species as deposited in the American Type Culture Collection (ATCC); Czech Collection of Microorganisms (CCM); Culture Collection, University of Göteborg, Sweden (CCUG); German Collection of Microorganisms and Cell Cultures (DSM); or Belgian Coordinated Collections of Microorganisms (LMG).

    • ↵ c Values represent total numbers of clinical isolates plus type and/or reference strain isolates.

  • TABLE 2.

    eap-targeting oligonucleotide primers used in this study

    NameOligonucleotide sequence (5′→3′)Amplicon size (bp)Source or reference
    EAP-CON1TAC TAA CGA AGC ATC TGC C230This study
    EAP-CON2TTA AAT CGA TAT CAC TAA TAC CTC
    EAP-P3wtGCA GCT AAG CCA TTA GAT AAA TCA TCA AGT TGC TTA CAC CVariableaDerived from primer P3 (19)b
    EAP-P2wtTTA AAA TTT AAT TTC AAT GTC TAC TTT TTT AAT GTC
    • ↵ a Strain-specific amplicon size depending on the number of tandem repeats in the eap gene.

    • ↵ b The primer sequences were modified by omitting the originally introduced restriction sites.

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eap Gene as Novel Target for Specific Identification of Staphylococcus aureus
Muzaffar Hussain, Christof von Eiff, Bhanu Sinha, Insa Joost, Mathias Herrmann, Georg Peters, Karsten Becker
Journal of Clinical Microbiology Feb 2008, 46 (2) 470-476; DOI: 10.1128/JCM.01425-07

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eap Gene as Novel Target for Specific Identification of Staphylococcus aureus
Muzaffar Hussain, Christof von Eiff, Bhanu Sinha, Insa Joost, Mathias Herrmann, Georg Peters, Karsten Becker
Journal of Clinical Microbiology Feb 2008, 46 (2) 470-476; DOI: 10.1128/JCM.01425-07
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KEYWORDS

Bacterial Proteins
polymerase chain reaction
RNA-Binding Proteins
Staphylococcal Infections
Staphylococcus aureus

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