, 2007). The spa types evolve by a combination of faster changes in the number of repeats and slower nucleotide point mutations (Brígido et al., 1991; Koreen et al., 2004). By slipped-strand mispairing during DNA replication (van Belkum, 1999), spa types seem more prone to deletions and duplications than to point mutations (Koreen et al., 2004; Kahl et al., 2005). During repeated subculturing, the repeat region of the spa gene proved to be very stable (Frénay et al., 1996), and sequencing of DNA from the same isolates in 10 different laboratories resulted in 100% reproducibility (Aires-de-Sousa et al., 2006). Few studies have addressed the question of how often and how fast spa types change in vivo in the patient or carrier

(Kahl et al., 2005; Kuhn et al., 2007; Sakwinska et al., 2010). Copenhagen is an area with low prevalence of methicillin-resistant

Staphylococcus aureus (MRSA) but with high variability of their spa types (Bartels et al., 2007). Since 2003, spa typing find more has been used in our department for outbreak investigation and identification of transmission routes of MRSA and to study the relationship between the different types. The aim of this study was to elucidate how often changes in the repeat region of the spa gene of MRSA occur over time, based on the analysis of repeated findings of MRSA from the same individual. The Department of Clinical Microbiology, Pirfenidone datasheet Hvidovre Hospital, services four of the five hospitals in Copenhagen and receives all microbiology samples from general practice in the Copenhagen and Frederiksberg Municipality (population 597 000). In the 5-year period 2003–2007, a total of 1843 MRSA isolates from 626 patients were spa typed (2003, 32; 2004, 137; 2005, 582; 2006, 662; 2007, 430). Of these patients, 307 had one isolate while 319 contributed with two or more MRSA isolates (1536 isolates in total from the 319 patients). The isolates

were from infections and carriage sites. ZD1839 order Staphylococcus aureus isolates were identified by positive Staphaurex (Remel Europe Ltd, Dartford, UK) and a positive coagulase test. Susceptibility testing was performed on Isosensi test agar by the disk-diffusion method (antibiotic disks; Oxoid, Basingstoke, UK) according to recommendations of the Swedish Reference Group for Antibiotics (http://www.srga.org). Isolates were screened for resistance to methicillin by a cefoxitin 10-μg disk. All MRSA isolates were confirmed mecA positive by PCR. Sequencing of the repeat region of the staphylococcal protein A gene (spa typing) was performed essentially as described (http://www3.ridom.de/staphtype/spa_sequencing.shtml). Sequence reactions were performed on both DNA strands and analyzed on an ABI Prism 3130XL (Applied Biosystems, Foster City, CA). For PCR and sequencing of the spa gene, primers 1113F (5′-taaagacgatccttcggtgagc-3′) and 1514R (5′-cagcagtagtgccgtttgctt-3′) were used. Designation of spa type was conducted using the ridomstaphtype software (Ridom GmbH, Würzburg, Germany) (Harmsen et al.