Lambda pfge ladder

Cell suspensions of four C. coli OR12 and aerotolerant derivatives were incorporated into Agarose plugs, lysed and washed as described by Ribot et al. (2001) (link). DNA plugs were cut into 2-mm slices and placed into 100 μl of 1x SuRE/Cut Buffer A (Roche) and equilibrated at 25°C for 15 min. This was replaced with 100 μl of 0.2 U/μl Sma I (Roche) in the same buffer and incubated for 2 h at 25°C. The plugs were incorporated in a gel consisting of 1% w/v Agarose (Bio-Rad) in TAE buffer. A 50–1000 kb DNA ladder (Lambda PFGE Ladder; New England BioLabs) was added as a marker. Electrophoresis was performed using a Bio-Rad CHEF-DRII. The gel was stained in 50 μg/ml ethidium bromide in TAE buffer and visualized under UV light using the Gel Doc XR system with the Quantity One basic software, version 4.6.5 (Bio-Rad).

Macrorestricton analysis using the enzyme ApaI (New England Biolabs, Frankfurt, Germany) and subsequent pulsed-field gel electrophoresis (PFGE) were performed for a preliminary characterisation of the 99 A. baumannii isolates as previously published [35 (link)], with a minor modification: for restriction analysis with ApaI (30U), the plug slices were incubated overnight at 25 °C. A Lambda PFGE ladder (New England Biolabs, Frankfurt, Germany) with a size range from 48.5 to 1018 kb served as size marker. Electrophoresis was performed using the CHEF-DR III system (Bio-Rad Laboratories, Düsseldorf, Germany). Gels were stained with GelRed (Biotium, San Francisco, CA, USA) and scanned with the laboratory’s imaging system (BIO RAD Molecular Imager GelDoc^TM XR+ with Image Lab^TM Software, Düsseldorf, Germany). An isolate from diagnostics (141_Diagnostik) served as internal control on each gel. Cluster analysis concerning the percentage similarity was performed with BioNumerics software, version 7.6.3 (Applied Maths, bioMérieux). Similarities were calculated with the dice coefficient (optimization 1.5%, tolerance 1.5%) and the unweighted pair group method with arithmetic mean (UPGMA) [35 (link)]. Pulsotypes were defined at a threshold value of ≥80% (named alphabetically) and at a threshold value of ≥87% (additional numeric marking) [35 (link),36 (link)].