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Journal of Clinical Microbiology, April 2003, p. 1499-1506, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1499-1506.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Influence of Transferable Genetic Determinants on the Outcome of Typing Methods Commonly Used for Enterococcus faecium

Guido Werner,1* Rob J. L. Willems,2 Bianca Hildebrandt,1 Ingo Klare,1 and Wolfgang Witte1

Robert Koch Institute, Wernigerode Branch, 38855 Wernigerode, Germany,1 National Institute of Public Health, Research Laboratory for Infectious Disease, 3721 Bilthoven, The Netherlands2

Received 1 August 2002/ Returned for modification 1 November 2002/ Accepted 26 December 2002

A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.

* Corresponding author. Mailing address: Robert Koch Institute, Wernigerode Branch, Burgstr. 37, 38855 Wernigerode, Germany. Phone: 0049 3943 679 210. Fax: 0049 3943 679 207. E-mail: wernerg{at}rki.de.

Journal of Clinical Microbiology, April 2003, p. 1499-1506, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1499-1506.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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