Cfr9i

DNA fingerprints of the MRSA isolates were illustrated by Cfr9I macro-restriction and PFGE [52 (link)]. Bacterial cells in agarose plugs were lysed by lysozyme, lysostaphin and detergents. After the plug-washing steps, chromosomal DNA was digested in 50 U of Cfr9I (Thermo Fisher Scientific, CA, USA). Macro-restriction fragments were separated in 1% agarose gel and TE buffer, with a switch time of 5–40 sec and 6 V/cm for 21 h, using CHEF-DRIII PFGE (Bio-Rad, CA, USA). The DNA band patterns were stained with ethidium bromide and documented under a UV illuminator. Relationships among strains were analysed by dendrogram construction using Dice coefficients, using the unweighted matrix pair group method with arithmetic mean (UPGMA) by Bionumeric software version 7.6 (Applied Maths, Belgium) with 1.5% optimization and 1.5% position tolerance. Pulsotypes were classified at >80% band pattern similarity, representing close clonal relatedness.

Suspensions were prepared from individual colonies after culture on Blood Columbia agar. OD at 600 nm was measured and suspensions were diluted to 10⁹ CFU/ml in EDTA-saline buffer (75 mmol/L NaCl and 25 mmol/L EDTA, pH 7.5), then mixed with an equal volume of 1% low-melting-point agarose and allowed to solidify in a 100 μL plug mould. The agarose plug was incubated for 24 h at 37°C in 500 μL lysis buffer (6 mmol/L Tris–HCl (pH 7.6), 0.1 mol/L EDTA, 1 mol/L NaCl, 0.5% Brij®58 (polyoxyethylene (20) cetyl ether; Sigma), 0.4% sodium deoxycholate, 0.5% sodium lauryl sarcosine and 1 mg/mL lysozyme (and 10 µg/mL lysostaphin to S. aureus)). The lysis buffer was replaced with 500 μL proteinase K buffer (1% sodium lauryl sarcosine, 0.5 mol/L EDTA (pH 9) and proteinase K (50 μg/mL; Sigma)) and this solution was incubated with gentle shaking at 50°C for 20 h. The plugs were then washed four times for 30 min at 37°C with 10 mL of Tris–EDTA buffer (10 mmol/L Tris–HCl (pH 8) and 1 mmol/L EDTA). One-third of a slice of each plug was cut and incubated for 18–20 h with 30 U of SpeI, or SmaI, XbaI and Cfr9I in the restriction buffer (Thermo scientific). DNA restriction fragments were separated in a PFGE apparatus at 14°C, 6 V/cm, for a specific time for each species (19–23 h). The gel was stained with gel-red and visualized with a UV system.