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: Background Ampicillin-resistant Enterococcus faecium isolates are reported in increasing numbers in many European hospitals. The clonal complex 17 (CC17) characterized by ampicillin resistance has been associated with nosocomial E. faecium outbreaks and infections in five continents. The aim was to investigate how prevalent ampicillin resistance is in clinical E. faecium isolates from Denmark and to investigate their clonal affiliation, especially to CC17. : Methods Microbiology data from 2002 through 2006 on E. faecium and Enterococcus faecalis blood isolates was received from Departments of Clinical Microbiology in 11 Danish counties. From January 2004 through December 2004, we collected 275 clinical enterococci from four of these departments. Multilocus sequence typing (MLST) and PFGE were performed on the 84 ampicillin-resistant E. faecium isolates from this collection. : Results A 68% increase in the number of infections caused by enterococci was observed from 2002 through 2006. The increase was mainly caused by E. faecium isolates, which tripled, whereas the number of E. faecalis isolates increased by only 23% during the same period. There was also a significant increase in the number of ampicillin-resistant E. faecium isolates. MLST showed that 98% of the tested ampicillin-resistant E. faecium isolates belonged to CC17. PFGE showed eight different clusters and we found indications of clonal spread within the hospitals. : Conclusions Ampicillin-resistant E. faecium isolates have increased in frequency in Denmark during 2002–2006. Most of the ampicillin-resistant E. faecium isolates belong to complex CC17. [ABSTRACT FROM AUTHOR]