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Journal of Clinical Microbiology, October 2004, p. 4473-4479, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4473-4479.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Multiplex PCR for the Detection of asa1, gelE, cylA, esp, and hyl Genes in Enterococci and Survey for Virulence Determinants among European Hospital Isolates of Enterococcus faecium

Vanessa Vankerckhoven,1* Tim Van Autgaerden,1 Carl Vael,1 Christine Lammens,1 Sabine Chapelle,1 Rosaria Rossi,2 Daniela Jabes,2 and Herman Goossens1,3

University of Antwerp, Antwerp, Belgium,1 Vicuron Pharmaceuticals, Gerenzano, Italy,2 University of Leiden, Leiden, The Netherlands3

Received 3 October 2003/ Returned for modification 7 January 2004/ Accepted 17 July 2004

A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

* Corresponding author. Mailing address: Laboratory of Medical Microbiology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Phone and fax: 32 3 820 26 63. E-mail: vanessa.vankerckhoven{at}ua.ac.be.

Journal of Clinical Microbiology, October 2004, p. 4473-4479, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4473-4479.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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