MRSA Identification and Molecular Characterization

Bacterial strains were cultured on blood agar at 35°C, after which extraction of DNA was performed by heat lysis. Briefly, a few colonies were suspended in molecular grade water and heated to 95°C for 15 minutes with shaking (300 rpm). After centrifugation at 14,500 rpm for 2 minutes, the supernatant was collected. Confirmation of MRSA was performed with a multiplex conventional PCR detecting the mecA gene, the S. aureus-specific spa gene, and the Panton-Valentine leukocidin (PVL) genes lukSF-PV, followed by gel electrophoresis [11 (link)]. For strains that were mecA negative, mecC PCR [5 (link)] was additionally performed.
All strains were spa-typed according to Harmsen et al. [11 (link)] with primers spa-1113f and spa-1514r [12 (link)] using the Ridom StaphType software and SpaServer [13 (link)]. The spa-types were assigned to known sequence types (ST) and/or clonal complexes (CC) based on Ridom Staphtyper [12 (link)] and pubMLST [14 (link), 15 (link)] databases. If a spa-type could not be assigned to a ST or CC, multi locus sequence typing (MLST) was performed, and CC was assigned using eBURST [16 (link)] software. A minimum spanning tree (MST) based on spa-repeats was constructed using Based Upon Repeat Pattern (BURP) clustering [17 (link)] with the Ridom SeqSphere + software [18 (link)]. To calculate genotypic diversity, Simpson’s diversity index [19 ] was used.