Gelcompar version 6

The SMC-associated hVISA/VISA strains were characterized by multilocus sequence typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec) typing, spa typing, agr typing, and pulsed-field gel electrophoresis (PFGE). MLST was carried out, and sequence types (STs) were assigned by reference to the S. aureus MLST website (http://saureus.mlst.net) [19 (link)].The SCCmec type was profiled by multiplex PCR assay [20 (link)]. The spa type was determined by PCR sequencing of the repeat region of the S. aureus protein, as described previously [21 (link)]. The agr specificity group was determined by PCR using specific primers as described previously [22 (link)]. Clonal relationships were determined by PFGE after DNA digestion by SmaI, as previously described [23 (link)]. PFGE patterns were analyzed with GelCompar version 6.6 (Applied Maths, Austin, Texas, USA) using the Dice coefficient and were represented by the unweighted pair-grouping method using arithmetic averages (UPGMA) with 1.7 tolerance and 1.2% optimization settings. Results were interpreted using a cut-off point of 80%.

DGGE profiling was applied to determine group-specific changes within the Bifidobacteria community. As each line in the fingerprint approximately represents one bacterial OTU, comparison of DGGE profiles, generated on samples at different timepoints throughout the experiment, allows the evaluation of qualitative changes in this group. After DNA extraction and PCR (7′ 95 °C; 35 × (1′ 94 °C/ 1′ 62 °C/2′ 72 °C); 10′ 72 °C 50–65%, 8%, 16 h, 38 V, 60 °C) with the Bifidobacteria group-specific primers, BIF164f and BIF662r [107 (link)], DGGE separated the PCR products. Gels were run using a DCodeTM Universal Mutation Detection System (Bio-Rad, Hercules, CA, USA), and data analysis was carried out using GelCompar, version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium). Pearson correlation and UMPG clustering were used to calculate dendrograms of DGGE profiles.